Genes dysregulated in autism as biomarkers and targets for therapeutic pathways

ABSTRACT

The disclosed invention comprises methods and materials for screening cells for genetic profiles associated with autism spectrum disorders. The methods typically involve isolating a cell from an individual and then observing the expression profile of one or more genes in the cell, wherein certain expression patterns of the genes observed are associated with autism spectrum disorders.

REFERENCE TO RELATED APPLICATIONS

This application claims priority under Section 119(e) from U.S. Provisional Application Ser. No. 61/489,471, filed May 24, 2011, the contents of which are incorporated herein by reference.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with Government support of Grant No. MH081754, awarded by the National Institutes of Health. The Government has certain rights in this invention.

FIELD OF THE INVENTION

The invention relates to methods and materials for observing gene expression profiles that are associated with conditions such as autism.

BACKGROUND OF THE INVENTION

Autism comprises a behaviorally defined spectrum of disorders characterized by impairment of social interaction, deficiency or abnormality of speech development, and limited activities and interest. To standardize the diagnosis of autism spectrum disorders (ASD), diagnostic criteria have been defined by the World Health Organization (International Classification of Diseases, 10th Revision (ICD-10), 1992) and the American Psychiatric Association (Diagnostic and Statistical Manual of Mental Disorders, 4th edition, Text Revision. Washington D.C., American Psychiatric Association, 2000 (DSM-IV)).

Genetic factors are significant determinants of autism spectrum disorders (see, e.g. Geschwind et al., (2007), Curr Opin Neurobiol, 17, 103-11). It has been shown that individuals with ASD carry chromosomal abnormality at a greater frequency than the general population (see, e.g. Veenstra-Vanderweele et al., (2004), Annu Rev Genomics Hum Genet, 5, 379-405; Vorstman et al., (2006), Mol Psychiatry, 11, 1, 18-28; Jacquemont et al. (2006), J Med Genet, 43, 843-9; Szatmari et al. (2007), Nat Genet, 39, 319-28; Sebat et al. (2007), Science, 316, 445-9). Maternally inherited duplication of 15q11-13 (dup15q) is the most common chromosomal abnormality in ASD. Over-expression of genes located in the duplicated region, including cytoplasmic FMR1 interacting protein 1 (CYFIPI), was shown in lymphoblastoid cell lines from ASD with dup15q (see, e.g. Nishimura et al. (2007), Hum Mol Genet, 16, 1682-98). A cryptic deletion located at the boundary of the first exon and first intron of ataxin-2 binding protein-1 (A2BP1) was identified in a female with ASD, resulting reduced mRNA expression in the individual's lymphocytes (see, e.g. Martin et al. (2007), Am J Med Genet B Neuropsychiatr Genet). Loss of copy number of neurexin 1 (NRXN1) was identified in two females sibs with ASD but not in either parent (see, e.g. Szatmari et al. (2007), Nat Genet, 39, 319-28). Loss of copy number and decreased expression of SH3 and multiple ankyrin repeat domains 3 (SHANK3) were identified in four individuals with ASD (see, e.g. Jeffries et al., (2005), Am J Med Genet A, 137, 139-47; and Durand et al. (2007), Nat Genet, 39, 25-7). A common ‘C’ allele in the promoter region of met proto-oncogene (MET) has also been shown to have a strong association with ASD (see, e.g. Campbell et al., (2006), Proc Natl Acad Sci USA, 103, 16834-9). The ‘C’ variant causes a twofold decrease in MET promoter activity. Such findings provide evidence that dysregulation of gene expression may affect susceptibility to and/or cause ASD.

Transcriptome profiling using DNA microarray represents an efficient manner in which to uncover an unanticipated relationship between gene expression alterations and neuropsychiatric diseases (see, e.g. Geschwind, D. H. (2003), Lancet Neurol, 2, 275-82; and Mimics et al., (2006), Biol Psychiatry, 60, 163-76). Several studies have suggested that blood-derived cells can be used to identify candidate genes in neuropsychiatric diseases, including ASD. Hu et al. analyzed gene expression profiling of lymphoblastoid cells from monozygotic (MZ) twins discordant in severity of ASD (see, e.g. Hu et al., (2006), BMC Genomics, 7, 118). Several genes were differentially expressed between MZ twins, suggesting candidate genes for ASD may be differentially expressed in lymphoblastoid cells from individuals with ASD. Previously analyses include genome-wide expression profiles of lymphoblastoid cells from ASD with full mutation of FMR1 (FMR1-FM) or dup15q, each of which account for 1-2% of ASD cases in large series, and non-autistic controls (see, e.g. Nishimura et al. (2007), Hum Mol Genet, 16, 1682-98). The gene expression profiles clearly distinguished ASD from controls and separated individuals with ASD based on their genetic etiology. The expression profiles also revealed shared pathways between ASD with FMR1-FM and ASD with dup15q.

While progress in understanding genetic factors associated with autism spectrum disorders has been made, specific assays for constellations of genetic factors associated with autism spectrum disorders would be a significant benefit to medical personnel. Tests for genetic factors associated with autism spectrum disorders are valuable for the diagnosis of this syndrome, as well as useful for research on the genetic mechanisms involved in autism spectrum disorders. Moreover, while there is no known medical treatment for autism, success has been reported for early intervention with behavioral therapies. In this context, such assays would facilitate the early identification of the disease, one now typically diagnosed between ages three and five. The increasing prevalence of autism spectrum disorders over recent years as well as the lack of effective means of genetic diagnosis, prevention or treatment make identifying ASD biomarkers and defining molecular pathways implicated in ASD a compelling goal.

SUMMARY OF THE INVENTION

Autism spectrum disorder is a heterogeneous condition and is likely to result from the combined effects of multiple, subtle genetic changes interacting with environmental factors. The disclosure provided herein characterizes genome-wide expression profiles of postmortem brain tissue from several brain regions in ASD patients and controls in order to identify genes showing consistent changes in mRNA levels in ASD brain. The ASD brain transcriptome is further analyzed using a network-based approach (co-expression network analysis), to identify groups of functionally related genes that are dysregulated at a transcriptional level in ASD brain. The experimental data presented herein highlights genes that are dysregulated at a transcriptional level in ASD in disease-relevant tissue and further defines sets of co-expressed genes that are useful as biomarkers for ASD, as well as being targets for therapeutic interventions.

The invention disclosed herein has a number of embodiments. Illustrative embodiments of the invention include methods of identifying a human cell having a gene expression profile associated with autism spectrum disorders by observing the expression of at least one gene in a test human cell, where the expression of that gene is observed to be dysregulated in individuals diagnosed with autism spectrum disorders (e.g. one or more of the genes disclosed in Tables A and B below). An illustrative embodiment of the invention is a method of identifying a test mammalian cell as having a gene expression profile observed in individuals diagnosed with autism by observing the expression of at least one gene comprising a sequence selected from the group consisting of SEQ ID NOs: 1-44 in the test mammalian cell in order to see if the test cell has a gene expression profile that is observed in individuals diagnosed with autism. In such methods, one can, for example, determine if levels of mRNA expression are at least two standard deviations away from levels of mRNA expression that are commonly observed in individuals not affected with autism. Alternatively in such methods, one can, for example, determine if the levels of mRNA expression are at least 20, 30, 40, 50, 60 or 70% above or below the levels of mRNA expression that are commonly observed in individuals not affected with autism.

In certain embodiments, methods of the invention are used to facilitate the diagnosis of an autism spectrum disorder. For example, in some embodiments of the invention, the cellular gene expression examined by such methods is that found in a test cell obtained from an individual identified as being predisposed to and/or exhibiting a behavior associated with autism spectrum disorders. Typically this cellular gene expression is compared to cellular gene expression in a control cell, for example, one obtained from an individual previously identified as not being predisposed to and/or exhibiting a behavior associated with autism spectrum disorders. In certain embodiments, the test cell examined by this method and the control cell are obtained from individuals who are related as siblings or as a parent and a child. Typically one or more cells used in these methods are leukocytes obtained from the peripheral blood.

In illustrative methods for observing an expression profile of one or more genes, mRNA expression is observed, for example by using a using quantitative PCR (qPCR) technique. In certain embodiments of the invention, the expression profile of the genes in is observed using a microarray of polynucleotides. Alternatively, polypeptide expression is observed and quantified, for example by using an antibody specific for a polypeptide encoded by a gene whose expression is shown to be dysregulated in autism spectrum disorders (e.g. using an ELISA technique or the like). Alternatively, the expression profile of a gene is observed using a single nucleotide polymorphism (SNP) detection or Southern blotting technique (e.g. to identify polymorphisms, deletions and/or duplications in genomic sequences).

Embodiments of the invention include kits comprising, for example, a first container, a label on said container, and a composition contained within said container; wherein the composition includes polymerase chain reaction (PCR) primer effective in the quantitative real time analysis of the mRNA expression levels of one or more genes disclosed herein whose expression is shown to be dysregulated in autism spectrum disorders (e.g. one or more of the 444 genes identified herein such as those disclosed in Table A or B); the label on said container, or a package insert included in said container indicates that the composition can be used to observe expression levels of these genes in at least one type of human leukocyte; a second container comprising a pharmaceutically-acceptable buffer; and instructions for using the PCR primer to obtain an expression profile of the one or more genes. Optionally the kit comprises 2, 3, 4, 5, 6, 7, 8, 9 or 10 polymerase chain reaction (PCR) primers effective in the quantitative real time analysis of the mRNA expression levels of different genes disclosed in the Tables below.

In some embodiments of the invention, one can observe an expression profile of at least, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more genes whose expression is shown to be dysregulated in autism spectrum disorders (e.g. using microarray technologies). In certain embodiments of the invention, the method is performed on a plurality of individuals and the results are then categorized based upon similarities or differences in their gene expression profiles. Optionally, the expression profile(s) is observed and/or collected and/or stored using a computer system comprising a processor element and a memory storage element adapted to process and store data from one or more expression profiles (e.g. in a library of such profiles). In this context, certain embodiments of the invention comprise an electronically searchable library of profiles, wherein the profiles include an individual's gene expression data in combination with other diagnostic data, for example assessments of behavior associated with autism spectrum disorders.

Other embodiments of this invention comprise methods of screening compounds that can modulate the mRNA and/or protein expression of a gene disclosed herein (e.g. those disclosed in Table A or B). Illustrative methods can include the steps of contacting a cell that expresses an endogenous or exogenous mRNA and/or protein with one or more test compounds and then determining if the one or more compounds modulates mRNA and/or protein expression in the cell (e.g. by qPCR techniques practiced on the cell in the presence and absence of the one or more compounds). A related embodiment of this invention comprises a method of screening compounds that interact with an mRNA or protein of a gene disclosure herein. Illustrative methods can include the steps of contacting one or more compounds with the mRNA or protein, and then determining if a compound interacts with the mRNA or protein (e.g. by binding techniques that separating compounds that interact with the mRNA or protein from compounds that do not).

Other objects, features and advantages of the present invention will become apparent to those skilled in the art from the following detailed description. It is to be understood, however, that the detailed description and specific examples, while indicating some embodiments of the present invention are given by way of illustration and not limitation. Many changes and modifications within the scope of the present invention may be made without departing from the spirit thereof, and the invention includes all such modifications.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a diagram depicting a number of genes showing significant expression differences between frontal and temporal cortex in control samples (top) and autism samples (bottom) at FDR <0.05 (left). Top 20 genes differentially expressed between frontal and temporal cortex in control samples (right). All of the genes shown are also differentially expressed between frontal and temporal cortex in fetal midgestation brain (see, e.g. Johnson, M. B. et al. Neuron 62, 494-509 (2009), but show no significant expression differences between frontal and temporal cortex in autism. The horizontal bars depict P values for differential expression between frontal and temporal cortex in the autism and control groups.

FIG. 2 shows A2BP1-dependent differential splicing events. (A) Top A2BP1-specific differential splicing events. Differential splicing events showing the most significant differences in alternative splicing between low-A2BP1 autism cases and controls as well as differential splicing differences consistent with the A2BP1 binding site position. The horizontal axis depicts the percentage of transcripts including the alternative exon. Lower Bar, autism samples; Top Bar, control samples. (B) Relevant gene ontology categories enriched in the set of genes containing exons differentially spliced between low-A2BP1 autism cases and controls.

FIG. 3 shows data illustrating the quantitative RT-PCR validation of DE genes. Average fold changes in the autism group (n>=5) relative to matched controls (n>=5) are shown (y-axis) for genes whose expression changes detected by arrays were validated by qRT-PCR. Top-upregulated genes, Bottom-downregulated genes. Error bars represent standard deviation of the mean.

FIG. 4 provides normalized expression values and ratios of temporal to frontal expression levels for selected genes showing attenuation of regional gene expression in ASD. FC•fold change, AF-ASD frontal cortex, AT-ASD temporal cortex, CF control frontal cortex, CT•control temporal cortex. Frontal and temporal cortex from the same brain are connected by a line.

FIG. 5 provides data showing A2BP1 expression values and A2BP1-dependent differential splicing events. (A) A2BP1 expression values as measured by microarrays. Expression values averages for two probes with highest expression level are plotted for both ASD and control groups. Black lines mark the mean and standard deviations from the mean. Smaller grey lines-Control samples used for RNA seq, “*”-ASD samples used for RNA seq. “**”-independent ASD samples used for RT PCR validation of DS events. (B) Semiquantatitive RT-PCR validation of DS events. A-pooled ASD samples (n=2-3), C-pooled control samples (n=2-3). Top panel: validation of DS events using the samples analyzed by RNA-seq. panel validation of DS events using additional independent ASD cases with low A2BP1 levels (shown as “*” in (A)).

FIGS. 6A-6I provide a Table showing GWAS p-values for genes in the M12 module, for which a SNP was mapped (see methods in the Example below) and the associated P-value was available (see, also Wang et al., Am. J. Hum. Genet. 81, 1278-1283 (2007) and/or Wang, K. et al. Nature 459, 528-533 (2009)).

FIG. 7 shows an embodiment of an illustrative computer system that can be used with embodiments of the invention.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined, all terms of art, notations and other scientific terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. The techniques and procedures described or referenced herein are generally well understood and commonly employed using conventional methodology by those skilled in the art. As appropriate, procedures involving the use of commercially available kits and reagents are generally carried out in accordance with manufacturer defined protocols and/or parameters unless otherwise noted.

Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims. It must also be noted that as used herein and in the appended claims, the singular forms “a”, “and”, and “the” include plural referents unless the context clearly dictates otherwise. All numbers recited in the specification and associated claims that refer to values that can be numerically characterized with a value other than a whole number (e.g. a number of standard deviations from a mean) are understood to be modified by the term “about”.

All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. Publications cited herein are cited for their disclosure prior to the filing date of the present application. Nothing here is to be construed as an admission that the inventors are not entitled to antedate the publications by virtue of an earlier priority date or prior date of invention. Further the actual publication dates may be different from those shown and require independent verification.

ILLUSTRATIVE EMBODIMENTS OF THE INVENTION

Autism is part of a spectrum of disorders including Asperger syndrome (AS) and other pervasive developmental disorders (PPD). The term “autism” is used herein according to its art accepted meaning and encompasses conditions of impaired social interaction and communication with restricted repetitive and stereotyped patterns of behavior, interests and activities present before the age of 3, to the extent that health may be impaired. AS is typically distinguished from other autistic disorders by a lack of a clinically significant delay in language development in the presence of the impaired social interaction and restricted repetitive behaviors, interests, and activities that characterize the autism-spectrum disorder (ASD). PPD-NOS (PPD, not otherwise specified) is typically used to categorize children who do not meet the strict criteria for autism but who come close, either by manifesting atypical autism or by nearly meeting the diagnostic criteria in two or three of the key areas.

The disclosure provided herein identifies genes that are observed to be Dysregulated in Autism Spectrum Disorders (e.g. autism as diagnosed by an Autism Diagnostic Interview (ADI-R), an Autism Diagnostic Observation Schedule (ADOS), an IQ surrogate test based on Raven's Progressive Matrices, observations of restricted repetitive behaviors or speech delay or the like). In the instant disclosure, these genes are collectively referred to as “DASD genes” for purposes of convenience. Human DASD genes useful in embodiments of the invention are shown for example in the Tables and Figures provided herein. Voineagu et al., Nature 474, 380-384 (2011) (the contents of which are incorporated by reference) includes illustrative information relating to these genes. Because the genes disclosed herein are known in the art and further because of the high level of skill possessed by artisans in this technical field, the information as disclosed herein places artisans in possession of the polynucleotide and polypeptide sequences of these genes by providing them with the specific disclosure which allows them to retrieve this sequence information from library sources such as GenBank and/or UniProtKB/Swiss-Prot with only minimal effort. As is know in the art, GenBank® is the NIH genetic sequence database, an annotated collection of all publicly available DNA sequences; and UniProtKB/Swiss-Prot is a curated protein sequence database which provides a high level of annotation (e.g. technical references describing the features of these genes), a minimal level of redundancy and high level of integration with other databases. The DASD gene polynucleotide and polypeptide sequence information can be retrieved from GenBank and/or UniProtKB/Swiss-Prot library databases by, for example, querying these databases using the DASD disclosure information as provided herein and/or incorporated by reference into the instant specification (e.g. the gene name, gene symbol, gene RefSeq number, gene locus etc.).

As disclosed in detail below, this disclosure provides methods and materials that can be used in the diagnosis and treatment of autism spectrum disorders, and autism-associated disorders. In typical embodiments of the invention one observes an expression profile of at least one gene disclosed herein, wherein a dysregulated expression profile provides evidence of an autism spectrum disorder. Embodiments of invention can be used for example in the diagnosis of (including a predisposition to), and/or treatment of autism spectrum disorders such as Asperger syndrome, pervasive developmental disorder, mental retardation, speech delay, and other associated psychiatric and neurological phenomena.

The invention disclosed herein has a number of embodiments. One embodiment is a method of identifying an individual having a gene expression profile associated with autism spectrum disorders comprising: observing an expression profile of one or more DASD genes in a test cell obtained from the individual (e.g. mRNA expression in a peripheral blood leukocyte obtained from an individual suspected of having a form of autism); wherein a determination that the expression of one (or more) DASD genes in the test cell exhibits a statistically significant difference from the expression of these DASD gene(s) as observed in control cell(s) (e.g. mRNA expression in a peripheral blood leukocyte obtained from a non-effected sibling) identifies the test cell as having a gene expression profile associated with autism spectrum disorders. In certain embodiments of the invention, the dysregulation of a group of genes and/or a specific pattern of changes in their expression (e.g. certain genes being overexpressed and other genes being under expressed) in such test cells as compared to control cells is used to characterize autism and/or identify individuals who have a high probability of having an ASD.

In typical embodiments of the invention, the gene expression profile comprises data relating to the levels of mRNA expressed by one or more DASD genes in the cell. In embodiments of the invention, gene expression can be qualified or quantified using a comparison of expression in a test cell relative to a mean expression observed in a control cell. For example, in some embodiments of the invention, mRNA expression levels of one or more DASD gene(s) in a test cell that is/are at least one, two, three, four or five standard deviations from the mean mRNA expression level(s) of these gene(s) as observed in control cell(s) identifies the test cell as having an expression profile associated with autism spectrum disorders. In other embodiments of the invention, mRNA expression levels of one or more DASD gene(s) in a test cell that is/are at least at least 20, 30, 40, 50, 60 or 70% above or below the mean mRNA expression level(s) of these gene(s) as observed in control cell(s) identifies the test cell as having an expression profile associated with autism spectrum disorders.

Typically in such methods of observing an expression profile of a DASD gene, mRNA expression is observed, for example by using a using quantitative PCR (qPCR) technique. In certain embodiments of the invention, the expression profile of the DASD gene in the test cell is observed using a microarray of polynucleotides. Alternatively, DASD polypeptide expression is observed, for example by using an antibody specific for a polypeptide encoded by a DASD gene (e.g. using an ELISA technique or the like). Alternatively, the expression profile is observed using Southern blotting (e.g. to identify deletions in or duplications of DASD genomic sequences).

Autism spectrum disorder is a heterogeneous condition that appears to result from the combined effects of multiple, subtle genetic changes interacting with environmental factors. Consequently, in some embodiments of the invention, an expression profile of at least, 2, 3, 4, 5, 6, 7, 8, 9 or 10, 15, 20, 25, 30, 35, 40 or more DASD genes are observed in order to obtain a detailed profile of these multiple genetic changes and/or to stratify individuals into subsets of autism spectrum disorders (e.g. using microarray technologies). For example, in certain embodiments of the invention, the method is performed on a plurality of individuals and then segregated based upon similarities or differences in their gene expression profiles. Optionally, the expression profile(s) of the test mammalian cell is observed using a computer system comprising a processor element and a memory storage element adapted to process and store data from one or more expression profiles (e.g. in a library of such profiles). In this context, one embodiment of the invention comprises an electronically searchable library of profiles, wherein the profiles include individual's gene expression data in combination with other diagnostic data, for example assessments of whether the individual exhibits behavior associated with an autism spectrum disorder (e.g. behavioral test data such as that obtained in an Autism Diagnostic Interview (ADI-R)).

In typical embodiments of the invention, these methods are used to facilitate diagnosis of an autism spectrum disorder in an individual. In this context, a cell examined in the methods of the invention can be a leukocyte obtained from the peripheral blood of the individual. In such cells, one can, for example examine the expression profile of one or more genes selected from the group consisting of: ACOT7, ALDH4A1, ATP1B1, ATP2B2, ATP6VOD₁, Clorf54, CD74, CEBPD, CFLAR, CIRBP, CMKOR1, CMTM7, CPNE3, DKFZP56400823, DNAJB1, ELMOD1, EMP3, FAM3C, FAM46A, G1P3, GNA12, HSPB1, ID3, IFITM2, IFITM3, INPPL1, ITGB5, ITPR1, JUN, LOC400566, LY96, LYPD1, MAP2K1, MCL1, MT2A, NEFM, NQ01, P4HA1, PITPNC1, PLEKHC1, PLOD2, PLTP, PREPL, PRKCB1, PTBP1, PTTGIIP, RHBDF2, SERTAD1, SLC29A1, SLC2A5, SOX9, STAMBPL1, TESC, TIMP1, TNFRSFIA, TNPO1, TXNIP, UCHL1, VAMP1, VHL, VIM, ZFP36, ZFP36L1. In some embodiments of the invention, the expression of all genes in this group are examined. In other embodiments of the invention, the expression of one or more of the genes in this group is not examined (e.g. by examining the expression of only 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or 20 or 30 or 40 etc. genes in this group). As is known in the art, individuals diagnosed with autism shown dysregulated gene expression in leukocytes (see, e.g. Nishimura et al., Human Molecular Genetics 2007 16(14): 1682-1698; and Hu et al., BMC Genomics 2006, 7: 1-18). Moreover, gene ontology enrichment analysis disclosed in the Example below showed that genes upregulated in autistic cortex were enriched for gene ontology categories implicated in immune and inflammatory response. Genes including those noted immediately above and/or identified in Table B were shown to have overlapping expression patterns with brain and blood cells in one or more data sets, confirming their utility as peripheral biomarkers.

In certain embodiments of the invention, the test cell is obtained from an individual previously identified as exhibiting a behavior associated with autism spectrum disorders. In some embodiments of the invention, the test cell is obtained from an individual identified as having a family member previously identified as exhibiting a behavior associated with autism spectrum disorders. In typical embodiments, the control mammalian cell is obtained from an individual previously identified as not exhibiting a behavior associated with autism spectrum disorders. Embodiments of the invention include methods which perform a further diagnostic procedure for autism spectrum disorders on an individual identified as having a gene expression profile associated with autism spectrum disorders (e.g. a procedure following standard validating measures, such as the Autism Diagnostic Interview (ADI-R)). Optionally, the test mammalian cell and the control mammalian cell are obtained from individuals who are related as siblings or as a parent and a child.

Embodiments of the invention further include a kit comprising: a first container, a label on said container, and a composition contained within said container; wherein the composition includes polymerase chain reaction (PCR) primer effective in the quantitative real time analysis of the mRNA expression levels of one or more DASD genes, the label on said container, or a package insert included in said container indicates that the composition can be used to observe expression levels of one or more DASD genes in at least one type of human leukocyte; a second container comprising a pharmaceutically-acceptable buffer; and instructions for using the PCR primer to obtain an expression profile of the one or more DASD genes. Optionally the kit comprises 2, 3, 4, 5, 6, 7, 8, 9 or 10 polymerase chain reaction (PCR) primers effective in the quantitative real time analysis of the mRNA expression levels of different DASD genes.

In certain embodiments of the invention, a kit further comprises a computer readable a memory storage element adapted to process and store data from one or more expression profiles. In some of these embodiments, the memory storage element organizes expression profile data into a format adapted for electronic comparisons with a library of expression profile data.

As noted above, embodiments of the invention compare DASD gene expression in a test cell (e.g. a cell obtained from an individual suspected of having an autism spectrum disorder) with DASD gene expression in a normal cell (e.g. a cell obtained from an individual not having an autism spectrum disorder) in order to determine if the test cell exhibits altered DASD gene expression. In addition to using normal cells as a comparative sample for DASD expression, in certain situations one can also use a predetermined normative value such as a predetermined normal sequence, and/or level of DASD mRNA or polypeptide expression (see, e.g., Greyer et al., J. Comp. Neurol. 1996 Dec. 9; 376(2):306-14 and U.S. Pat. No. 5,837,501) to evaluate levels of DASD expression in a given sample. The term “status” in this context is used according to its art accepted meaning and refers to the condition or state of a gene and its products. Typically, skilled artisans use a number of parameters to evaluate the condition or state of a gene and its products. These include, but are not limited to the level, sequence of and biological activity of expressed gene products (such as DASD mRNA, polynucleotides and polypeptides). In certain embodiments of the invention, the expression of a DASD gene product is characterized by observing how far the expression level of a DASD mRNA in a sample deviates from a mean expression level of that mRNA in control cells in order to obtain a statistical measure of precision. Standard deviation is a measure of the variability or dispersion of a data set, in this case, the levels of mRNA expression of selected genes. Standard deviation in this context allows determinations of how spread out a set of expression values is and how a given sample fits into such analyses. Illustrative statistical methods for determining such values can be found for example in Cui et al., Genome Biol. (2003) 4:210; Tusher et al., Proc. Natl. Acad. Sci. USA (2001) 98:5116-5121; Jeffery et al., BMC Bioinformatics (2006) 7:359; and Breitling et al., FEBS Lett. (2004) 573:83-92, the contents of which are incorporated by reference.

As discussed in detail below, the status of a DASD gene can be analyzed by a number of techniques that are well known in the art. Typical protocols for evaluating the status of the DASD gene and gene products are found, for example in Ausubel et al. eds., 1995, Current Protocols In Molecular Biology, Units 2 (Northern Blotting), 4 (Southern Blotting), 15 (Immunoblotting) and 18 (PCR Analysis). The status of a DASD gene in a biological sample is evaluated by various methods utilized by skilled artisans including, but not limited to single nucleotide polymorphism analyses and genomic Southern analysis (to examine, for example perturbations in DASD genomic sequences), Northern analysis and/or PCR analysis of DASD mRNA (to examine, for example alterations in the polynucleotide sequences or expression levels of DASD mRNAs), and, Western and/or immunohistochemical analysis (to examine, for example alterations in polypeptide sequences, alterations in expression levels of DASD proteins etc.). Detectable DASD polynucleotides include, for example, a DASD gene or fragment thereof, DASD mRNA, alternative splice variants, DASD mRNAs, and recombinant DNA or RNA molecules comprising a DASD polynucleotide.

By examining a biological sample obtained from an individual (e.g. a peripheral blood leukocyte) for evidence altered gene expression of one or more genes whose expression is dysregulated in individuals diagnosed with autism spectrum disorders, medical personnel can obtain information useful in the identification, treatment and/or management of these disorders. Typically, the methods comprise detecting in a sample from a subject the presence of altered DASD gene expression, the presence of the alteration being indicative of the presence of, or predisposition to autism, an autism spectrum disorder, or an autism-associated disorder. In this context, “altered gene expression” encompasses altered DASD mRNA and/or polypeptide levels; altered DASD polynucleotide and polypeptide sequences, altered DASD genomic DNA methylation patterns and the like, alterations that are typically absent in individuals not having an autism spectrum disorder. In such examinations, the status of one or more DASD polynucleotides and/or polypeptides in a biological sample of interest (e.g. a peripheral blood leukocyte obtained from an individual suspected of having an autism spectrum disorder) can be compared to a standard or control, for example, or to the status of the DASD polynucleotide(s) or polypeptide(s) in a corresponding normal sample (e.g. a peripheral blood leukocyte obtained from a non-effected sibling or another individual not having a autism spectrum disorder). An alteration in the status of DASD gene expression in the biological sample (as compared to a control or standardized sample and/or value) then provides evidence of an autism spectrum disorder.

As noted above, embodiments of invention provide methods that comprise for example observing the expression status of one or more DASD genes in a subject in order to obtain diagnostically and/or prognostically useful information. Such methods typically use a leukocyte obtained from a subject to assess the status of a DASD gene. However, the sample may be a variety of biological samples derived from a subject, which contains nucleic acids or polypeptides. Examples of such samples include fluids, tissues, cell samples, organs, biopsies, etc. Most preferred samples are blood and other leukocyte containing tissues etc. Pre-natal diagnosis may also be performed by testing for example fetal cells or placental cells. Other biological samples from which DASD genes and/or the products of DASD genes can be isolated is suitable. The sample may be collected according to conventional techniques and used directly for diagnosis or stored. The sample may be treated prior to performing the method, in order to render or improve availability of nucleic acids and/or polypeptides for testing. Treatments include, for example, lysis (e.g., mechanical, physical, chemical, etc.), centrifugation, etc. Also, the nucleic acids and/or polypeptides may be pre-purified or enriched by conventional techniques, and/or reduced in complexity. Nucleic acids and polypeptides may also be treated with enzymes or other chemical or physical treatments to produce fragments thereof.

The isolation of biological samples from a subject which contain nucleic acids and/or polypeptides is well know in the art. For example, certain embodiments isolate leukocytes from the circulating blood in order to assess the status of DASD genes in these cells. In such embodiments, blood is typically collected from subjects into heparinized blood collection tubes by personnel trained in phlebotomy using sterile technique. The collected blood samples can be divided into aliquots and centrifuged, and the buffy coat layer can then be removed (this fraction contains the leukocytes). RNA can then be extracted using a commercial RNA purification kit (e.g. RNeasy; Qiagen, Valencia, Calif.). RNA quality can be determined, for example, with an A260/A280 ratio and capillary electrophoresis on an apparatus such as an Agilent 2100 Bioanalyzer automated analysis system (Agilent Technologies, Palo Alto, Calif.).

In typical embodiments of the invention, a sample is contacted with reagents such as probes, primers or ligands (e.g. antibodies) in order to assess the presence of altered gene expression of a DASD gene. Such methods may be performed by a wide variety of apparatuses used in the art, such as a plate, tube, well, glass, etc. In specific embodiments, the contacting is performed on a substrate coated with the reagent, such as a nucleic acid array or a specific ligand (e.g. antibody) array. The substrate may be solid or semi-solid substrate such as any support comprising glass, plastic, nylon, paper, metal, polymers and the like. The substrate may be of various forms and sizes, such as a chip, a slide, a membrane, a bead, a column, a gel, etc. The contacting may be made under any condition suitable for a complex to be formed between the reagent and the nucleic acids or polypeptides of the sample.

A wide variety of methods known in the art can be used to examine the expression of DASD polypeptides and polynucleotides in cells such as peripheral blood leukocytes. For example, certain embodiments of methods which examine DASD polynucleotides and polypeptides in such cells are analogous to those methods from well-established diagnostic assays known in the art such as those that observe the expression of biomarkers such as prostate specific antigen (PSA) polynucleotides and polypeptides. For example, just as PSA polynucleotides are used as probes (for example in Northern analysis, see, e.g., Sharief et al., Biochem. Mol. Biol. Int. 33(3):567-74 (1994)) and primers (for example in PCR analysis, see, e.g., Okegawa et al., J. Urol. 163(4): 1189-1190 (2000)) to observe the presence and/or the level of PSA mRNAs in methods of monitoring PSA expression, the DASD polynucleotides identified herein can be utilized in the same way to observe DASD overexpression or underexpression or other alterations in these genes. Similarly, just as PSA polypeptides are used to generate antibodies specific for PSA which can then be used to observe the presence and/or the level of PSA proteins in methods to monitor PSA protein expression (see, e.g., Stephan et al., Urology 55(4):560-3 (2000)) in prostate cells (see, e.g., Alanen et al., Pathol. Res. Pract. 192(3):233-7 (1996)), the DASD polypeptides described herein can be utilized to generate antibodies for use in detecting DASD expression in peripheral blood leukocytes and the like. Accordingly, the status of DASD gene products provides information useful for predicting a variety of factors including the presence of and/or susceptibility to autism spectrum disorders. As discussed in detail herein, the status of DASD gene products in patient samples can be analyzed by a variety protocols that are well known in the art including immunohistochemical analysis, the variety of Northern blotting techniques including in situ hybridization, RT-PCR analysis (e.g. quantitative RT-PCR), Western blot analysis, polynucleotide and polypeptide microarray analysis and the like.

Exemplary embodiments of the invention include methods for identifying a cell that overexpresses or underexpresses DASD polynucleotides and/or polypeptides. One such embodiment of the invention is an assay that quantifies the expression of the DASD gene in a cell by detecting the absence/presence and/or relative levels of DASD mRNA concentrations in the cell. Methods for the evaluation of particular mRNAs in cells are well known and include, for example, hybridization assays using complementary DNA probes (such as in situ hybridization using labeled DASD riboprobes, Northern blot and related techniques) and various nucleic acid amplification assays (such as qPCR using complementary primers specific for DASD, and other amplification type detection methods, such as, for example, branched DNA, SISBA, TMA and the like).

Embodiments of the invention include methods for detecting a DASD mRNA in a biological sample by generating cDNA in the sample by reverse transcription using at least one primer; amplifying the cDNA so produced using an DASD polynucleotides as sense and antisense primers to amplify DASD cDNAs therein; and detecting the presence of the amplified DASD cDNA. One exemplary PCR method that can be used in embodiments of the invention is a real-time quantitative PCR (qPCR) assay. Such real-time assays provide a large dynamic range of detection and a highly sensitive methods for determining the amount of DNA template of interest. When qPCR follows a reverse transcription reaction, it can be used to quantify RNA templates as well. In addition, qPCR makes quantification of DNA and RNA much more precise and reproducible because it relies on the analysis of PCR kinetics rather than endpoint measurements. Illustrative qPCR assays are disclosed for example in U.S. Patent Application Nos.: 2006/0008809; 2003/0219788; 2006/0051787; and 2006/0099620, the contents of which are incorporated by reference.

Some embodiments of the invention can use next-generation sequencing technologies for the expression profiling of DASD genes, for example those that are commercially available from vendors such as APPLIED BIOSYSTEMS and ILLUMINA (e.g. ILLUMINA Ref8 v3 microarrays). Typically in these embodiments, one can count the number of copies of each DASD gene that is expressed in order to provide assays that quantify the expression levels of all mRNA molecules in a cell. Because such methods are based on sequencing and not hybridization, they can provide an unbiased, probe-less measurement of all mRNA molecules in a sample. Illustrative aspects of such technologies are disclosed for example in U.S. Patent Application Publication No. 20080262747, the contents of which are incorporated by reference.

Another embodiment of the invention is a method of detecting DASD genes having altered copy numbers (i.e. genes having a copy numbers that is above or below the number of copies observed in cells obtained from normal individuals) and/or another chromosomal rearrangement in a biological sample by isolating genomic DNA from the sample; amplifying the isolated genomic DNA using DASD polynucleotides as sense and antisense primers; and detecting the presence of the altered DASD gene. Any number of appropriate sense and antisense probe combinations can be designed from the nucleotide sequence provided for the DASD and used for this purpose.

The invention also provides assays for detecting the presence of a DASD protein in a tissue or other biological sample and the like. Methods for detecting a DASD-related protein are also well known and include, for example, immunoprecipitation, immunohistochemical analysis, Western blot analysis, molecular binding assays, ELISA, ELIFA and the like. For example, a method of detecting the presence of a DASD-related protein in a biological sample comprises first contacting the sample with a DASD antibody, a DASD-reactive fragment thereof, or a recombinant protein containing an antigen binding region of a DASD antibody; and then detecting the binding of DASD-related protein in the sample. Optionally, DASD polypeptide expression is measured in a tissue microarray.

In another embodiment of the invention, one can evaluate the status DASD nucleotide and amino acid sequences in a biological sample in order to identify perturbations in the structure of these molecules. These perturbations can include insertions, deletions, substitutions, duplications and the like in the coding and regulatory regions of the DASD gene. Such evaluations are useful because perturbations in the nucleotide and amino acid sequences are observed in a large number of proteins associated with a growth dysregulated phenotype (see, e.g., Marrogi et al., 1999, J. Cutan. Pathol. 26(8):369-378). For example, a mutation in the sequence of an DASD 5′ or 3′ regulatory enhancer and/or promoter sequence may provide evidence of dysregulated expression. Such assays therefore have diagnostic and predictive value where a mutation in DASD is indicative of dysregulated expression.

A wide variety of assays for observing perturbations in nucleotide and amino acid sequences are well known in the art. For example, the size and structure of nucleic acid or amino acid sequences of DASD gene products are observed by the Northern, Southern, Western, PCR and DNA sequencing protocols discussed herein. In addition, other methods for observing perturbations in nucleotide and amino acid sequences such as single strand conformation polymorphism analysis are well known in the art (see, e.g., U.S. Pat. Nos. 5,382,510 and 5,952,170, the contents of which are incorporated by reference).

The mutation in a DASD gene may be a single base substitution mutation resulting in an amino acid substitution, a single base substitution mutation resulting in a translational stop, an insertion mutation, a deletion mutation, or a gene rearrangement. The mutation may be located in an intron, an exon of the gene, or a promotor or other regulatory region which affects the expression of the gene. Screening for mutated nucleic acids can be accomplished by direct sequencing of nucleic acids. Nucleic acid sequences can be determined through a number of different techniques which are well known to those skilled in the art, for example by chemical or enzymatic methods. The enzymatic methods rely on the ability of DNA polymerase to extend a primer, hybridized to the template to be sequenced, until a chain-terminating nucleotide is incorporated. The most common methods utilize dideoxynucleotides. Primers may be labelled with radioactive or fluorescent labels. Various DNA polymerases are available including Klenow fragment, AMV reverse transcriptase, Thermus aquaticus DNA polymerase, and modified T7 polymerase.

Ligase chain reaction (LCR) is yet another method of screening for mutated nucleic acids. LCR can be carried out in accordance with known techniques and is especially useful to amplify, and thereby detect, single nucleotide differences between two DNA samples. In general, the reaction is carried out with two pairs of oligonucleotide probes: one pair binds to one strand of the sequence to be detected; the other pair binds to the other strand of the sequence to be detected. The reaction is carried out by, first, denaturing (e.g., separating) the strands of the sequence to be detected, then reacting the strands with the two pairs of oligonucleotide probes in the presence of a heat stable ligase so that each pair of oligonucleotide probes hybridize to target DNA and, if there is perfect complementarity at their junction, adjacent probes are ligated together. The hybridized molecules are then separated under denaturation conditions. The process is cyclically repeated until the sequence has been amplified to the desired degree. Detection may then be carried out in a manner like that described above with respect to PCR.

Southern hybridization is also an effective method of identifying differences in sequences. Hybridization conditions, such as salt concentration and temperature can be adjusted for the sequence to be screened. Southern blotting and hybridizations protocols are described in Current Protocols in Molecular Biology (Greene Publishing Associates and Wiley-Interscience), pages 2.9.1-2.9.10. Probes can be labelled for hybridization with random oligomers (primarily 9-mers) and the Klenow fragment of DNA polymerase. Very high specific activity probe can be obtained using commercially available kits such as the Ready-To-Go DNA Labelling Beads (Pharmacia Biotech), following the manufacturer's protocol. Briefly, 25 ng of DNA (probe) is labelled with ³²P-dCTP in a 15 minute incubation at 37° C. Labelled probe is then purified over a ChromaSpin (Clontech) nucleic acid purification column.

Determinations of the presence of the polymorphic form of a DASD protein can also be carried out, for example, by isoelectric focusing, protein sizing, or immunoassay. In an immunoassay, an antibody that selectively binds to the mutated protein can be utilized (for example, an antibody that selectively binds to the mutated form of DASD encoded protein). Such methods for isoelectric focusing and immunoassay are well known in the art. For example, changes resulting in amino acid substitutions, where the substituted amino acid has a different charge than the original amino acid, can be detected by isoelectric focusing. Isoelectric focusing of the polypeptide through a gel having an ampholine gradient at high voltages separates proteins by their pI. The pH gradient gel can be compared to a simultaneously run gel containing the wild-type protein. Protein sizing techniques such as protein electrophoresis and sizing chromatography can also be used to detect changes in the size of the product.

As an alternative to isoelectric focusing or protein sizing, the step of determining the presence of the mutated polypeptides in a sample may be carried out by an antibody assay with an antibody which selectively binds to the mutated polypeptides (i.e., an antibody which binds to the mutated polypeptides but exhibits essentially no binding to the wild-type polypeptide without the polymorphism in the same binding conditions). Antibodies used to selectively bind the products of the mutated genes can be produced by any suitable technique. For example, monoclonal antibodies may be produced in a hybridoma cell line according to the techniques of Kohler and Milstein, Nature, 265, 495 (1975), which is hereby incorporated by reference. A hybridoma is an immortalized cell line which is capable of secreting a specific monoclonal antibody. The mutated products of genes which are associated with autism may be obtained from a human patient, purified, and used as the immunogen for the production of monoclonal or polyclonal antibodies. Purified polypeptides may be produced by recombinant means to express a biologically active isoform, or even an immunogenic fragment thereof may be used as an immunogen. Monoclonal Fab fragments may be produced in Escherichia coli from the known sequences by recombinant techniques known to those skilled in the art.

Additionally, one can examine the methylation status of the DASD gene in a biological sample. Aberrant demethylation and/or hypermethylation of CpG islands in gene 5′ regulatory regions frequently occurs in immortalized and transformed cells, and can result in altered expression of various genes. For example, promoter hypermethylation of the pi-class glutathione S-transferase (a protein expressed in normal prostate but not expressed in >90% of prostate carcinomas) appears to permanently silence transcription of this gene and is the most frequently detected genomic alteration in prostate carcinomas (De Marzo et al., Am. J. Pathol. 155(6): 1985-1992 (1999)). A variety of assays for examining methylation status of a gene are well known in the art. For example, one can utilize, in Southern hybridization approaches, methylation-sensitive restriction enzymes which cannot cleave sequences that contain methylated CpG sites to assess the methylation status of CpG islands. In addition, MSP (methylation specific PCR) can rapidly profile the methylation status of all the CpG sites present in a CpG island of a given gene. This procedure involves initial modification of DNA by sodium bisulfite (which will convert all unmethylated cytosines to uracil) followed by amplification using primers specific for methylated versus unmethylated DNA. Protocols involving methylation interference can also be found for example in Current Protocols In Molecular Biology, Unit 12, Frederick M. Ausubel et al. eds., 1995.

Embodiments of the invention include compositions that can be used for example in various methods disclosed herein. Compositions useful in the methods disclosed herein typically include for example one or more DASD nucleic acid molecules designed for use as a probe such as a PCR primer in a method used to monitor DASD mRNAs or genomic sequences in a cell. Optionally, the probe or primer has 8, 9, 19, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more nucleotides that are complementary to a DASD mRNA. In certain embodiments, the probe or primer comprises 5-25 heterologous polynucleotide sequences (e.g. to facilitate cloning). Typically, the probe or primer will hybridize to the DASD mRNA under “stringent conditions” i.e. those that: (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (3) employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC (sodium chloride/sodium. citrate) and 50% formamide at 55° C., followed by a high-stringency wash consisting of 0.1×SSC containing EDTA at 55° C.

Specifically contemplated nucleic acid related embodiments of the invention disclosed herein are genomic DNA, cDNAs, ribozymes, and antisense molecules, as well as nucleic acid molecules based on an alternative backbone, or including alternative bases, whether derived from natural sources or synthesized, and include molecules capable of inhibiting the RNA or protein expression of DASD. For example, antisense molecules can be RNAs or other molecules, including peptide nucleic acids (PNAs) or non-nucleic acid molecules such as phosphorothioate derivatives, that specifically bind DNA or RNA in a base pair-dependent manner. Compositions of the invention include one or more antibodies that bind DASD and which can be used as a probe to monitor DASD polypeptide expression in a cell. A skilled artisan can readily prepare these polynucleotide and polypeptide compounds using the DASD polynucleotides and polynucleotide sequences and associated information that is disclosed herein.

For use in the methods described above, kits are also provided by the invention. Such kits may comprise a carrier means being compartmentalized to receive in close confinement one or more container means such as vials, tubes, and the like, each of the container means comprising one of the separate elements to be used in the method. For example, one of the container means may comprise a probe that is or can be detectably labeled. Such probe may be an antibody or polynucleotide specific for DASD protein or DASD gene or message, respectively. Where the kit utilizes nucleic acid hybridization to detect the target nucleic acid, the kit may also have containers containing nucleotide(s) for amplification of the target nucleic acid sequence and/or a container comprising a reporter-means, such as a biotin-binding protein, such as avidin or streptavidin, bound to a reporter molecule, such as an enzymatic, florescent, or radioisotope label.

The kits of the invention have a number of embodiments. A typical embodiment is a kit comprising a container, a label on the container, and a composition contained within the container; wherein the composition includes: (1) a polynucleotide that hybridizes to a complement of the DASD polynucleotide and/or (2) an antibody that binds the DASD polypeptide, the label on the container indicates that the composition can be used to evaluate the expression level of the DASD gene product in at least one type of mammalian cell (e.g. a human peripheral blood leukocyte), and instructions for using the DASD polynucleotide or antibody for evaluating the presence of DASD RNA, DNA or protein in at least one type of mammalian cell.

Autism is a heterogeneous condition and is likely to result from the combined effects of multiple, genetic changes including copy number variations and single nucleotide polymorphisms, interacting with environmental factors (see, e.g. Folstein et al., (2001) Nat. Rev. Genet., 2, 943-955; Belmonte et al., (2004) Mol. Psychiatry, 9, 646-663; Veenstra-Vanderweele et al., (2004) Annu Rev Genomics Hum Genet, 5, 379-405; and Muhle et al., (2004) Pediatrics, 113, e472-486). Classifications such as a computer based hierarchy of autistic patients based on genotypic and phenotypic information is one effective way to identify more homogeneous subgroups and hasten the identification of genes underlying autism (see, e.g. Folstein et al., (2001) Nat. Rev. Genet., 2, 943-955; Belmonte et al., (2004) Mol. Psychiatry, 9, 646-663; Veenstra-Vanderweele et al., (2004) Annu Rev Genomics Hum Genet, 5, 379-405; and Muhle et al., (2004) Pediatrics, 113, e472-486). About 3% of autistic children have either FMR1-FM or dup(15 q), thus comprising more homogeneous populations with a single major genetic etiology for their autism.

In this context, embodiments of the invention further provides methods of obtaining a gene expression profile associated with autism spectrum disorders and methods of generating a database, or collection, of such profiles. The methods generally involve observing a gene expression profile associated with autism spectrum disorders, storing the data on a computer readable medium (CRM), and linking the data with at least one additional data point such as an individual identifying code and/or familial genetic information and/or the presence or absence of other phenomena (e.g. behavioral phenomena) associated with autism spectrum disorders such as Asperger syndrome, pervasive developmental disorder, mental retardation, speech delay, and other associated psychiatric and neurological phenomena. The profile having this information is then recorded on a CRM.

Computer related embodiments of the invention disclosed herein can be performed for example, using one of the many computer systems known in the art. For example, embodiments of the invention can include a searchable database library comprising a plurality of cell profiles recorded on a computer readable medium, each of the profiles comprising further information such as identifying codes and/or familial relationships and/or gene expression and/or behavioral phenomena associated with autism spectrum disorders. In this context, one can then use this library of gene expression and behavioral data to, for example, classify and/or examine etiological subsets of autism as well as to explore the pathophysiology of this condition. In one embodiment of the invention, data obtained from a new test sample is compared to data in such a library in order to, for example, find similar comparative profiles in the library from which diagnostic and/or prognostic information can be inferred. FIG. 7 illustrates an exemplary generalized computer system 202 that can be used to implement elements of the present invention. The computer 202 typically comprises a general purpose hardware processor 204A and/or a special purpose hardware processor 204B (hereinafter alternatively collectively referred to as processor 204) and a memory 206, such as random access memory (RAM). The computer 202 may be coupled to other devices, including input/output (I/O) devices such as a keyboard 214, a mouse device 216 and a printer 228.

In one embodiment, the computer 202 operates by the general purpose processor 204A performing instructions defined by the computer program 210 under control of an operating system 208. The computer program 210 and/or the operating system 208 may be stored in the memory 206 and may interface with the user and/or other devices to accept input and commands and, based on such input and commands and the instructions defined by the computer program 210 and operating system 208 to provide output and results. Output/results may be presented on the display 222 or provided to another device for presentation or further processing or action. In one embodiment, the display 222 comprises a liquid crystal display (LCD) having a plurality of separately addressable liquid crystals. Each liquid crystal of the display 222 changes to an opaque or translucent state to form a part of the image on the display in response to the data or information generated by the processor 204 from the application of the instructions of the computer program 210 and/or operating system 208 to the input and commands. The image may be provided through a graphical user interface (GUI) module 218A. Although the GUI module 218A is depicted as a separate module, the instructions performing the GUI functions can be resident or distributed in the operating system 208, the computer program 210, or implemented with special purpose memory and processors.

Some or all of the operations performed by the computer 202 according to the computer program 210 instructions may be implemented in a special purpose processor 204B. In this embodiment, some or all of the computer program 210 instructions may be implemented via firmware instructions stored in a read only memory (ROM), a programmable read only memory (PROM) or flash memory in within the special purpose processor 204B or in memory 206. The special purpose processor 204B may also be hardwired through circuit design to perform some or all of the operations to implement the present invention. Further, the special purpose processor 204B may be a hybrid processor, which includes dedicated circuitry for performing a subset of functions, and other circuits for performing more general functions such as responding to computer program instructions. In one embodiment, the special purpose processor is an application specific integrated circuit (ASIC).

The computer 202 may also implement a compiler 212 which allows an application program 210 written in a programming language such as COBOL, C++, FORTRAN, or other language to be translated into processor 204 readable code. After completion, the application or computer program 210 accesses and manipulates data accepted from I/O devices and stored in the memory 206 of the computer 202 using the relationships and logic that was generated using the compiler 212. The computer 202 also optionally comprises an external communication device such as a modem, satellite link, Ethernet card, or other device for accepting input from and providing output to other computers.

In one embodiment, instructions implementing the operating system 208, the computer program 210, and the compiler 212 are tangibly embodied in a computer-readable medium, e.g., data storage device 220, which could include one or more fixed or removable data storage devices, such as a zip drive, floppy disc drive 224, hard drive, CD-ROM drive, tape drive, etc. Further, the operating system 208 and the computer program 210 are comprised of computer program instructions which, when accessed, read and executed by the computer 202, causes the computer 202 to perform the steps necessary to implement and/or use the present invention or to load the program of instructions into a memory, thus creating a special purpose data structure causing the computer to operate as a specially programmed computer executing the method steps described herein. Computer program 210 and/or operating instructions may also be tangibly embodied in memory 206 and/or data communications devices 230, thereby making a computer program product or article of manufacture according to the invention. As such, the terms “article of manufacture,” “program storage device,” and “computer program product” as used herein are intended to encompass a computer program accessible from any computer readable device or media.

Of course, those skilled in the art will recognize that any combination of the above components, or any number of different components, peripherals, and other devices, may be used with the computer 202. Although the term “user computer” is referred to herein, it is understood that a user computer 102 may include portable devices such as medication infusion pumps, analyte sensing apparatuses, cellphones, notebook computers, pocket computers, or any other device with suitable processing, communication, and input/output capability.

Embodiments of the invention further comprise, for example, methods of assessing the response of a subject to a treatment of an autism spectrum disorder, or an autism-associated disorder (e.g. treatment comprising the administration of a therapeutic agent), the method comprising detecting altered DASD gene or polypeptide expression (e.g. in multiple genes selected from the group consisting of those whose polynucleotide sequences are shown in SEQ ID NOs: 1-44) in a sample from the treated subject, the presence of the alteration being indicative of a response to the treatment. One embodiment of this invention comprises a method of screening for a compound that modulates DASD mRNA and/or protein expression comprising the steps of contacting a cell that expresses an endogenous or exogenous DASD mRNA and/or protein with one or more compounds and then determining if the one or more compounds modulates DASD mRNA and/or protein expression in the cell (e.g. by qPCR techniques practiced on the cell in the presence and absence of the one or more compounds). Optionally the method comprises observing an effect of a compound on an expression profile of at least one gene comprising a sequence selected from the group consisting of SEQ ID NOs: 1-44, the method comprising the steps of observing an expression profile of the at least one gene in the presence of the compound; and then comparing the expression profile that is observed in the presence of the compound with the expression profile that is observed in the absence of the compound, so that the effect of the compound on an expression profile of the at least one gene is observed.

Another embodiment of this invention comprises a method of screening for a compound that interacts with one or more DASD mRNAs or proteins comprising the steps of contacting one or more compounds with the DASD mRNA and/or protein, and then determining if a compound interacts with the DASD mRNA and/or protein (e.g. by binding techniques that separating compounds that interact with the DASD mRNA and/or protein from compounds that do not). This embodiment of the invention can be used for example to screen chemical libraries for compounds which modulate, e.g., inhibit, antagonize, or agonize or mimic, the expression of a DASD gene as measured by one of the assays disclosed herein. The chemical libraries can be peptide libraries, peptidomimetic libraries, chemically synthesized libraries, recombinant, e.g., phage display libraries, and in vitro translation-based libraries, other non-peptide synthetic organic libraries. Exemplary libraries are commercially available from several sources (e.g. e, Tripos/PanLabs, ChemDesign, Pharmacopoeia). Typical peptide libraries and screening methods that can be used to identify compounds that modulate the expression of and/or interact with DASD protein sequences are disclosed for example in U.S. Pat. Nos. 5,723,286 and 5,733,731, the contents of which are incorporated by reference.

Various aspects of the invention are further described and illustrated by way of the examples that follow, none of which are intended to limit the scope of the invention. Certain disclosure in the examples below can be found in Voineagu et al., Nature 474, 380-384 (2011), the contents of which are incorporated by reference. The disclosure in the Voineagu et al. Nature article is found in U.S. Provisional Application Ser. No. 61/489,471, filed May 24, 2011 (the priority document for the instant application). This Voineagu et al. Nature article provides information that illustrates aspects of the technologies described herein. In addition, certain methods and materials that can be adapted for use with embodiments of the invention can be those found for example in U.S. Patent Application Nos.: 2002/0155450; 2006/0141519; 2007/0134664; and 2009/0011414, the contents of which are incorporated by reference.

EXAMPLES Example 1 Transcriptomic Analysis of Autistic Brain Reveals Convergent Molecular Pathology

Autism spectrum disorder (ASD) is a common, highly heritable neurodevelopmental condition characterized by marked genetic heterogeneity (see references 1-3, which, like all of the other numerically identified references in this Example, are listed below). Thus, a fundamental question is whether autism represents an etiologically heterogeneous disorder in which the myriad genetic or environmental risk factors perturb common underlying molecular pathways in the brain (4). Here, we demonstrate consistent differences in transcriptome organization between autistic and normal brain by gene co-expression network analysis. Remarkably, regional patterns of gene expression that typically distinguish frontal and temporal cortex are significantly attenuated in the ASD brain, suggesting abnormalities in cortical patterning. We further identify discrete modules of co-expressed genes associated with autism: a neuronal module enriched for known autism susceptibility genes, including the neuronal specific splicing factor A2BP1 (also known as RBFOX1), and a module enriched for immune genes and glial markers. Using high throughput RNA sequencing we demonstrate dysregulated splicing of A2BP1-dependent alternative exons in the ASD brain. Moreover, using a published autism genome-wide association study (GWAS) data set, we show that the neuronal module is enriched for genetically associated variants, providing independent support for the causal involvement of these genes in autism. In contrast, the immune-glial module showed no enrichment for autism GWAS signals, indicating a non-genetic etiology for this process. Collectively, our results provide strong evidence for convergent molecular abnormalities in ASD, and implicate transcriptional and splicing dysregulation as underlying mechanisms of neuronal dysfunction in this disorder.

We analyzed post-mortem brain tissue samples from 19 autism cases and 17 controls from the Autism Tissue Project and the Harvard brain bank (see, e.g., supplementary Table 1 in Voineagu et al., Nature 474, 380-384 (2011) using ILLUMINA microarrays. For each individual, we profiled three regions previously implicated in autism (5): superior temporal gyrus (STG, also known as Brodmann's area (BA) 41/42), prefrontal cortex (BA9) and cerebellar vermis. After filtering for high-quality array data (see Methods below), we retained 58 cortex samples (29 autism, 29 controls) and 21 cerebellum samples (11 autism, 10 controls) for further analysis (see Methods for detailed sample description). We identified 444 genes showing significant expression changes in autism cortex samples (DS1, see, e.g., FIG. 1 b in Voineagu et al., Nature 474, 380-384 (2011)), and only 2 genes were differentially expressed between the autism and control groups in cerebellum (Methods), indicating that gene expression changes associated with autism were more pronounced in the cerebral cortex, which became the focus of further analysis (see, e.g., supplementary Table 2 in Voineagu et al., Nature 474, 380-384 (2011)). There was no significant difference in age, post mortem interval (PMI), or RNA integrity numbers (RIN) between autism and control cortex samples (see, e.g., supplementary FIG. 1, in Voineagu et al., Nature 474, 380-384 (2011)).

Supervised hierarchical clustering based on the top 200 differentially expressed genes showed distinct clustering of the majority of autism cortex samples (see, e.g., FIG. 1 a in Voineagu et al., Nature 474, 380-384 (2011)), including one case that was simultaneously found to have a 15q duplication (see, e.g., supplementary Table 1 in Voineagu et al., Nature 474, 380-384 (2011)), which is known to cause 1% of ASD (6). Cortex samples from ten of the cases coalesced in a single tight-clustering branch of the dendrogram. Clustering was independent of age, sex, RIN, PMI, co-morbidity of seizures, or medication (see, e.g., FIG. 1 a and Supplementary FIG. 2 c in Voineagu et al., Nature 474, 380-384 (2011)). It is interesting to note that the two ASD cases that cluster with controls (see, e.g., FIG. 1 a in Voineagu et al., Nature 474, 380-384 (2011)) are the least severe cases, as assessed by global functioning (see, e.g., supplementary Table 12 in Voineagu et al., Nature 474, 380-384 (2011)). We observed a highly significant overlap between differentially expressed genes in frontal and temporal cortex (P=10⁻⁴⁴; see, e.g., FIG. 1 b in Voineagu et al., Nature 474, 380-384 (2011)), supporting the robustness of the data and indicating that the autism-specific expression changes are consistent across these cortical areas. We also validated a cross section of the differentially expressed genes by quantitative reverse transcription PCR (RT-PCR) and confirmed microarray-predicted changes in 83% of the genes tested (see, e.g., FIG. 3 herein and supplementary FIG. 2 b in Voineagu et al., Nature 474, 380-384 (2011)). Gene ontology enrichment analysis (Methods) showed that the 209 genes downregulated in autistic cortex were enriched for gene ontology categories related to synaptic function, whereas the upregulated genes (N=235) showed enrichment for gene ontology categories implicated in immune and inflammatory response (see, e.g., supplementary Table 3 in Voineagu et al., Nature 474, 380-384 (2011)).

To test whether these findings were replicable, and to further validate the results in an independent data set, we obtained tissue from an additional frontal cortex region (BA44/45) from nine ASD cases and five controls (DS2; see, e.g. supplementary Table 4 in Voineagu et al., Nature 474, 380-384 (2011)). Three of the cases and all of the controls used for validation were independent from our initial cohort. Ninety-seven genes were differentially expressed in BA44/45 in DS2, and 81 of these were also differentially expressed in our initial cohort (P=1.2×10⁻⁹³, hypergeometric test; see, e.g., FIG. 1 b, c in Voineagu et al., Nature 474, 380-384 (2011)). Remarkably, the direction of expression differences between autism and controls was the same as in the initial cohort for all but 2 of the 81 overlapping differentially expressed probes. Hierarchical clustering of DS2 samples based on either the top 200 genes differentially expressed in the initial cohort or the 81 overlapping genes showed distinct separation of cases from controls (see, e.g., supplementary FIG. 6 in Voineagu et al., Nature 474, 380-384 (2011)). In addition, comparison of these differentially expressed results with another, smaller study of the STG in ASD (7), revealed significant consistency at the level of differentially expressed genes, including downregulation of DLX1 and AHI1 (see, e.g., supplementary Table 5 in Voineagu et al., Nature 474, 380-384 (2011)). Thus, differential expression analysis produced robust and highly reproducible results, warranting further refined analysis.

We next applied weighted-gene co-expression network analysis (WGCNA) (8,9) to integrate the expression differences observed between autistic and control cerebral cortex into a higher order, systems level context. We first asked whether there are global differences in the organization of the brain transcriptome between autistic and control brain by constructing separate co-expression networks for the autism and control groups (Methods). The control brain network showed high similarity with the previously described human brain co-expression networks (see, e.g., supplementary Table 7 in Voineagu et al., Nature 474, 380-384 (2011)), consistent with the existence of robust modules of co-expressed genes related to specific cell types and biological functions (8). Similarly, the majority of the autism modules (87%) showed significant overlap with the previously described human brain modules (see, e.g., supplementary Table 6 in Voineagu et al., Nature 474, 380-384 (2011)), indicating that many features reflecting the general organization of the autism brain transcriptome are consistent with that of the normal human brain.

The expression levels of each module were summarized by the first principal component (the module eigengene), and were used to assess whether modules are related to clinical phenotypes or other experimental variables, such as brain region. Two of the control module eigengenes (cM6, cM13) showed significant differences (P<0.05) between the two cortical regions as expected, whereas none of the ASD modules showed any differences between frontal and temporal cortex. This led us to explore the hypothesis that the normal molecular distinctions between the two cortical regions tested were altered in ASD compared with controls. Remarkably, whereas 174 genes were differentially expressed between control BA9 and BA41 (false discovery rate (FDR)<1%), none of the genes were differentially expressed in the same regional comparison among the ASD cases. This was not simply an issue of statistical thresholds, as relaxing the statistical criteria for differential expression to an FDR of 5% identified over 500 differentially expressed genes in controls, and only 8 in ASD brains, confirming the large difference observed in regional cortical differential gene expression between ASD cases and controls (see, e.g., FIG. 1 herein and FIG. 1 d, in Voineagu et al., Nature 474, 380-384 (2011)). Analysis of differential expression from a data set (10) of gene expression in developing fetal human brain showed a highly significant (P=5.8×10⁻⁹) overlap of differentially expressed genes with those found in controls in this study, independently confirming that these genes differentiate normal temporal and frontal lobes. We evaluated the homogeneity of gene expression variance across the autism and control groups using Bartlett's test (Methods) which indicated that increased variance was not the major factor responsible for the striking difference in regional gene expression between ASD and controls (see, e.g., FIG. 4 herein and supplementary FIG. 7 and Supplementary Data in Voineagu et al., Nature 474, 380-384 (2011)).

These data suggest that typical regional differences, many of which are observed during fetal development (10), are attenuated in frontal and temporal lobe in autism brain, pointing to abnormal developmental patterning as a potential pathophysiological driver in ASD. This is especially interesting in light of a recent anatomical study of five cases with adult autism which demonstrated a reduction in typical ultra structural differences between three frontal cortical regions in autism (11). Together, these independent studies provide both molecular and structural evidence suggesting a relative diminution of cortical regional identity in autism.

To identify discrete groups of co-expressed genes showing transcriptional differences between autism and controls, we constructed a coexpression network using the entire data set, composed of both autism and control samples (Methods). As previously shown for complex diseases (12,13) co-expression networks allow analysis of gene expression variation related to multiple disease-related and genetic traits. We assessed module eigengene relationship to autism disease status, age, gender, cause of death, co-morbidity of seizures, family history of psychiatric disease, and medication, providing a complementary assessment of these potential confounders to that performed in the standard differential expression analysis (see, e.g., supplementary Table 9 in Voineagu et al., Nature 474, 380-384 (2011)).

The comparison between autism and control groups revealed two network modules whose eigengenes were highly correlated with disease status, and not any of the potential confounding variables (see, e.g., supplementary Table 9 in Voineagu et al., Nature 474, 380-384 (2011)). We found that the top module (M12) showed highly significant enrichment for neuronal markers (see, e.g., supplementary Table 9 in Voineagu et al., Nature 474, 380-384 (2011)), and high overlap with two neuronal modules previously identified as part of the human brain transcriptional network (8): a PVALB+ interneuron module and a module of genes involved in synaptic function. The M12 eigengene was under-expressed in autism cases, indicating that genes in this module were downregulated in the autistic brain (see, e.g., FIG. 2 in Voineagu et al., Nature 474, 380-384 (2011)). Consistent with the pathways identified to be downregulated in autism by differential expression analysis (see, e.g., supplementary Table 3 in Voineagu et al., Nature 474, 380-384 (2011)), the functional enrichment of M12 included the gene ontology categories involved in synaptic function, vesicular transport and neuronal projection.

Remarkably, unlike differentially expressed genes, M12 showed significant overrepresentation of known autism susceptibility genes (2) (see, e.g., supplementary Table 10 in Voineagu et al., Nature 474, 380-384 (2011); P=6.1×10⁻⁴), including CADPS2, AHI1, CNTNAP2, and SLC25A12, supporting the increased power of the network-based approach to identify disease-relevant transcriptional changes. A further advantage of network analysis over standard analysis of differential expression is that it allows one to infer the functional relevance of genes based on their network position (9). The hubs of M12, that is, the genes with the highest rank of M12 membership8, were A2BP1, APBA2, SCAMP5, CNTNAP1, KLC2, and CHRM1 (see, e.g., supplementary Data in Voineagu et al., Nature 474, 380-384 (2011)). The first three of these genes have previously been implicated in autism (14-16), whereas the fourth is a homologue of the autism susceptibility gene CNTNAP2 (17). We contemplate the group of genes most strongly connected to the known ASD genes (see, e.g., supplementary FIG. 5 in Voineagu et al., Nature 474, 380-384 (2011)) and emphasize the downregulation of several interneuron markers, such as DLX1 and PVALB, as candidates for future genetic and pathologic investigations.

The second module of co-expressed genes highly related to autism disease status, M16, was enriched for astrocyte markers and markers of activated microglia (see, e.g., supplementary Table 9 in Voineagu et al., Nature 474, 380-384 (2011)), as well as for genes belonging to immune and inflammatory gene ontology categories (see, e.g., FIG. 2 in Voineagu et al., Nature 474, 380-384 (2011)). This module, which was upregulated in ASD brain, showed significant similarity to two modules identified in previous studies of normal human brain (8): an astrocyte module and a microglial module. Consistent with this functional annotation, two of the hubs of the M16 module were known astrocyte markers (ADFP, also known as PLIN2, and IFITM2).

One of the hubs of the M12 module was A2BP1, a neural- and muscle specific alternative splicing regulator (18) and the only splicing factor previously implicated in ASD (16). Because A2BP1 was downregulated in several ASD cases (see, e.g., FIG. 5 herein and supplementary FIG. 8 in Voineagu et al., Nature 474, 380-384 (2011)), this observation provided a unique opportunity to identify potential disease-relevant A2BP1 targets. Whereas A2BP1-regulated alternative exons have been predicted genome-wide (19), few genes have been experimentally validated as A2BP1 targets (20). To identify potential A2BP1-dependent differential splicing events in ASD brain, we performed high-throughput RNA sequencing (RNA-Seq) on three autism samples with significant downregulation of A2BP1 (average fold change by quantitative RT-PCR=5.9) and three control samples with average A2BP1 levels. We identified 212 significant alternative splicing events (see, e.g., supplementary Data in Voineagu et al., Nature 474, 380-384 (2011)). Among these, 36 had been defined (19) as predicted targets of A2BP1/2, which represents a highly significant overlap (P=2.2×10⁻¹⁶). In addition, five previously validated A2BP1 targets showed evidence of alternative splicing, four of which (ATP5C1, ATP2B1, GRIN1 and MEF2C) were confirmed as having differential splicing between ASD samples with low A2BP1 expression and control samples, indicating that we were able to identify a high proportion of the expected A2BP1-dependent differential splicing events. We also observe that alternative exons with increased skipping in ASD relative to control cases are significantly enriched for A2BP1 motifs in adjacent, downstream intronic sequences (P=1.09×10⁻⁷, Fisher's exact test), consistent with previous data (19).

The top gene ontology categories enriched among ASD differential splicing genes highly overlapped with the gene ontology categories found to be enriched in the M12 module (see, e.g., FIG. 2 herein and FIG. 3 b in Voineagu et al., Nature 474, 380-384 (2011)). In addition, A2BP1 target genes showed enrichment for actin-binding proteins and genes involved in cytoskeleton reorganization (see, e.g., FIG. 2 herein and FIG. 3 b in Voineagu et al., Nature 474, 380-384 (2011)). Among top predicted A2BP1-dependent differential splicing events (see, e.g., FIG. 2 herein and FIG. 3 a in Voineagu et al., Nature 474, 380-384 (2011)) are CAMK2G, which also belongs to the M12 module, as well as NRCAM and GRIN1. The latter are proteins involved in synaptogenesis, in which allelic variants have been associated with autism and schizophrenia, respectively (21,22).

RT-PCR assays confirmed a high proportion (85%) of the tested differential splicing changes involving predicted A2BP1 targets (see, e.g., FIG. 5 herein and supplementary FIG. 8 in Voineagu et al., Nature 474, 380-384 (2011)). We further tested the differential splicing events validated by RT-PCR in three independent ASD cases with decreased A2BP1 levels and confirmed the predicted changes in alternative splicing (see, e.g., FIG. 5 herein and supplementary FIG. 8 in Voineagu et al., Nature 474, 380-384 (2011)), indicating that the observed differential splicing events are indeed associated with reduced A2BP1 levels, rather than due to inter-individual variability. The RNA-Seq data thus provides validation of the functional groups of genes identified by coexpression analysis, and evidence for a convergence of transcriptional and alternative-splicing abnormalities in the synaptic and signalling pathogenesis of ASD.

To test whether our findings are more generalizable, and determine whether the autism-associated transcriptional differences observed are likely to be causal, versus collateral effects or environmentally induced changes, we tested whether our co-expression modules or the differentially expressed genes show enrichment for autism genetic association signals. M12 showed highly significant enrichment for association signals (P=5×10⁻⁴), but neither M16 nor the list of differentially expressed genes showed such enrichment (see, e.g., FIG. 4 in Voineagu et al., Nature 474, 380-384 (2011)). As a negative control, we performed the same set-enrichment analysis using two GWAS studies for non-psychiatric disease performed on the same genotyping platform: a genome-wide association for hair colour (23), and a GWAS study of warfarin maintenance dose (24) finding no significant enrichment of the association signal (see, e.g., FIG. 4 b, Supplementary FIG. 4 in Voineagu et al., Nature 474, 380-384 (2011)). These results indicate that (1) M12 consists of a set of genes that are supported by independent lines of evidence to be causally involved in ASD pathophysiology, and (2) the upregulation of immune response genes in the autistic brain observed by us and others (25) has no evidence of a common genetic component.

Our system-level analysis of the ASD brain transcriptome demonstrates the existence of convergent molecular abnormalities in ASD for the first time, providing a molecular neuropathological basis for the disease, whose genetic, epigenetic, or environmental etiologies can now be directly explored. The genome-wide analysis performed here significantly extends previous findings implicating synaptic dysfunction, as well as microglial and immune dysregulation in ASD (6) by providing an unbiased systematic assessment of transcriptional alterations and their genetic basis. We show that the transcriptome changes observed in ASD brain converge with GWAS data in supporting the genetic basis of synaptic and neuronal signalling dysfunction in ASD, whereas immune changes have a less pronounced genetic component and thus are most likely either secondary phenomena or caused by environmental factors. Because immune molecules and cells such as microglia have a role in synaptic development and function (26), we speculate that the observed immune upregulation may be related to abnormal ongoing plasticity in the ASD brain. The striking attenuation of gene expression differences observed here between frontal and temporal cortex in ASD is likely to represent a defect of developmental patterning and provides a strong rationale for further studies to assess the pervasiveness of transcriptional patterning abnormalities across the ASD brain. We also demonstrate for the first time alterations in differential splicing associated with A2BP1 levels in the ASD brain, and show that many of the affected exons belong to genes involved in synaptic function. Finally, given current evidence of genetic overlap between ASD and other neurodevelopmental disorders including schizophrenia and attention deficit hyperactivity disorder (ADHD), our data provide a new pathway-based framework from which to assess the enrichment of genetic association signals in other psychiatric disorders.

Methods Brain Tissue.

Post-mortem brain tissue was obtained from the Autism Tissue Project and the Harvard Brain Bank as well as the MRC London Brain bank for Neurodegenerative Disease. Brain tissue samples from 19 autism cases and 17 controls were obtained from the Autism Tissue Project (ATP) and the Harvard Brain Bank. For each brain, tissue was obtained from frontal cortex (BA9), temporal cortex (BA41/42 or BA22) and cerebellum (vermis), with the exception of three controls lacking the cerebellum sample (see, e.g., supplementary Table 1 in Voineagu et al., Nature 474, 380-384 (2011)). For the replication experiment, frontal cortex tissue (BA44/45) from nine ASD cases and five controls were obtained from the ATP and MRCLondon Brain bank for Neurodegenerative Disease respectively (see, e.g., supplementary Table 4 in Voineagu et al., Nature 474, 380-384 (2011)).

For all of the autism cases, clinical information is available upon request from ATP (see www.autismtissueprogram.org), including the ADI-R diagnostic scores. Supplementary Table 12 in Voineagu et al., Nature 474, 380-384 (2011) contains a summary of clinical characteristics. Although autism cases with known genetic causes were not included in this study, one case with a chromosome 15q duplication was identified for AN17138 by high density small nucleotide polymorphism (SNP) arrays (28) during the course of this study. The ATP cases were genotyped with high-density SNP arrays and with two exceptions all are Caucasians. The two Asian samples cluster with the other ASD cases in the current study, and are not distinguishable from the Caucasian cases based on clustering by gene expression.

Microarrays and RNA-Seq.

Total RNA was extracted from 100 mg of tissue using a Qiagen miRNA kit according to the manufacturer's protocol. Expression profiles were obtained using ILLUMINA Ref8 v3 microarrays. RNA-seq was performed on the ILLUMINA GAIIx, as per the manufacturer's instructions. Further detailed information on data analysis is available in Methods. All microarray and RNA-seq data are deposited in GEO under accession number GSE28521.

RNA Extractions and Microarrays.

Total RNA was extracted from approximately 100 mg of frozen tissue, using the Qiagen miRNA kit. RNA concentration was assessed by a NanoDrop and RNA quality was measured using an Agilent Bioanalyzer. All RNA samples included in the expression analysis had an RNA integrity number (RIN)>5. cDNA labelling and hybridizations on ILLUMINA Ref8 v3 microarrays were performed according to the manufacturer's protocol.

Microarray Data Analysis.

Microarray data analysis was performed using the R software and Bioconductor packages. Raw expression data were log 2 transformed and normalized by quantile normalization. Data quality control criteria included high inter-array correlation (Pearson correlation coefficients >0.85) and detection of outlier arrays based on mean inter-array correlation and hierarchical clustering. Probes were considered robustly expressed if the detection P value was <0.05 for at least half of the samples in the data set. Cortex samples (58: 29 autism, 29 controls) and cerebellum samples (21: 11 autism, 10 controls) fulfilled all data quality control criteria. The 29 autism cortex samples included tissue from 13 ASD cases with both frontal and temporal cortex and 3 ASD cases with frontal cortex only (in total 16 frontal cortex and 13 temporal cortex ASD samples). The 29 autism control samples also included tissue from 13 controls with both frontal and temporal cortex and 3 controls with frontal cortex only (in total 16 frontal cortex and 13 temporal cortex control samples).

Initially, all samples were normalized together to assess clustering by brain region. As expected, we observed distinct clustering of cortex and cerebellum samples (see, e.g., supplementary FIG. 2A in Voineagu et al., Nature 474, 380-384 (2011)). For subsequent analyses, cortex samples and cerebellum samples were normalized and analyzed separately.

Differential Expression.

Differential expression was assessed using the SAM package (significance analysis of microarrays, see www-stat.stanford.edu/,tibs/SAM) and unless otherwise specified the significance threshold was FDR<0.05 and fold changes >1.3. Given that SAM is less sensitive in detecting differentially expressed genes for small number of samples, for the replication cohort, the differential expression was assessed by a linear regression method (Limma package, see bioconductor.org/packages/release/bioc/html/limma.html). Our results showing high degree of overlap between genes differentially expressed in the two data sets indicate that the expression differences observed are independent of the analysis methods.

Because 444 genes were differentially expressed between autism and controls in cortex and only 2 genes were differentially expressed between the two groups in cerebellum (FDR<0.05), we tested whether this difference was due to the smaller number of cerebellum samples, by relaxing the statistical criteria to FDR<0.25. We found fewer than 10 differentially expressed genes in cerebellum using the relaxed statistical criteria, supporting the conclusion that genome-wide expression changes in autism were more pronounced in cerebral cortex than in cerebellum.

To account for the fact that the control group of DS1 contained samples from a single female whereas the autism DS1 group included four females, we eliminated from differential expression analysis all probes showing evidence of gender specific gene expression (n=70). We also applied linear regression of expression values against age and sex, and then assessed differential expression between the autism and control groups using the residual values. We observed a 96% overlap between differentially expressed genes using either the residual values or the raw data, indicating that neither age nor sex were major drivers of expression differences between the autism and control groups.

Differential expression between frontal and temporal cortex was assessed by a paired modified t-test (SAM) using the 13 autism and 13 control cases for which RNA samples from both cortex areas passed the quality control criteria. For each of the 510 genes that were differentially expressed in control samples between frontal and temporal cortex, we compared the variance of autism and control expression values in frontal cortex and temporal cortex. The homogeneity of variance (homoscedasticity) of gene expression was assessed using the Barlett test in R. Fifty one genes showed a significant difference in variance (P<0.05, Barlett test) between autism and control groups both in frontal and temporal cortex, and the Barlett test P-values for these genes are listed in Supplementary Data in Voineagu et al., Nature 474, 380-384 (2011).

WGCNA.

Unsigned co-expression networks were built using the WGCNA package in R. Probes with evidence of robust expression (9,914; see above) were included in the network. Network construction was performed using the blockwise Modules function in the WGCNA package (29), which allows the network construction for the entire data set. For each set of genes a pair-wise correlation matrix is computed, and an adjacency matrix is calculated by raising the correlation matrix to a power. The power of 10 was chosen using the scale-free topology criterion (9) and was used for all three networks: the network built using autism samples only, controls samples only or all samples. An advantage of weighted correlation networks is the fact that the results are highly robust with respect to the choice of the power parameter. For each pair of genes, a robust measure of network interconnectedness (topological overlap measure) was calculated based on the adjacency matrix. The topological overlap based dissimilarity was then used as input for average linkage hierarchical clustering. Finally, modules were defined as branches of the resulting clustering tree. To cut the branches, we used the hybrid dynamic tree-cutting because it leads to robustly defined modules (31). To obtain moderately large and distinct modules, we set the minimum module size to 40 genes and the minimum height for merging modules at 0.1. Each module was summarized by the first principal component of the scaled (standardized) module expression profiles. Thus, the module eigengene explains the maximum amount of variation of the module expression levels. For each module, we defined the module membership measure (also known as module eigengene based connectivity kME) as the correlation between gene expression values and the module eigengene. Genes were assigned to a module if they had a high module membership to the module (kME.0.7). An advantage of this definition (and the kME measure) is that it allows genes to be part of more than one module. Genes that did not fulfill these criteria for any of the modules are assigned to the grey module. For the cell type marker enrichment analysis we used the markers defined experimentally in refs (32) and (33) which were previously used to annotate human brain network modules (34,35).

Module visualization: the topological overlap measure was calculated for the top 100 genes in each module ranked by kME. The resulting list of gene pairs was filtered so that both genes in a pair had the highest kME for the module plotted (that is, most module-specific interactions). The resulting top 150 gene pairs were plotted using Visant.

Gene Ontology Analyses.

Functional enrichment was assessed using the DAVID database see david.abcc.ncifcrf.gov/. For differentially expressed genes and coexpression modules, the background was set to the total list of genes expressed in the brain in the cortex data set. For genes containing differentially spliced exons, the background was set to the total set of genes showing evidence of alternative splicing in our RNA-seq data. The statistical significance threshold level for all gene ontology enrichment analyses was P<0.05 (Benjamin and Hochberg corrected for multiple comparisons).

Statistical Analyses.

All gene set overlap analyses were performed by assessing cumulative hypergeometric probability using the phyper function in R. The population size was defined as the total number of probes expressed in both data sets. If the comparison involved different platforms, the comparison was done at gene level.

Quantitative RT-PCR.

One microgram of total RNA was treated with RNase free DNase I (INVITROGEN/Fermentas) and reverse-transcribed using INVITROGEN Superscript II reverse-transcriptase and random hexanucleotide primers (INVITROGEN). Real time PCR was performed on an ABI7900 cycler in 10 ml volume containing iTaq Sybrgreen (BIORAD) and primers at a concentration of 0.5 mM each. The results shown in FIG. 3 (see also supplementary FIG. 2 b in Voineagu et al., Nature 474, 380-384 (2011)) represent at least two independent cDNA synthesis experiments for each gene. GAPDH levels were used as an internal control. Statistical significance was assessed by a two-tailed t-test assuming unequal variance.

Semi-Quantitative RT-PCR.

Total RNA (600 ng) pooled from autism cases (n=2-3) or controls (n=2-3) was reverse-transcribed as described above. cDNA (50 ng) was subjected to 30 cycles of PCR amplification using the primers described in Supplementary Table 11 in Voineagu et al., Nature 474, 380-384 (2011). PCR products were separated on a 3% agarose gel stained with GELSTAR (LONZA).

RNA Sequencing and Data Analysis.

73-nucleotide reads were generated using an ILLUMINA GAIT sequencer according to the manufacturer's protocol. To generate sufficient read coverage for the quantitative analysis of alternative splicing events, reads for ASD and control brain samples were separately pooled and aligned to an existing database of EST and cDNA-derived alternative splicing junctions using the Basic Local Alignment Tool (BLAT) as described previously (36,37). Reads were considered properly aligned to a splice junction if at least 71 of the 73 nucleotides matched and at least 5 nucleotides mapped to each of the two exons forming the splice junction. Alternative exon inclusion values (“% inc”), representing the proportion of messenger RNA transcripts with the alternatively spliced exon included, were calculated for each mRNA pool as the ratio of reads aligning to the C1-A or A-C2 junctions against reads aligning against all three possible junctions as previously described (36) (C1-A, A-C2, C₁-C₂, see, e.g. supplementary FIG. 3 in Voineagu et al., Nature 474, 380-384 (2011).). Calculated % inc values were considered reliable if at least one of the included junctions as well as the skipped junctions were covered by at least 20 reads. % inc values were compared across samples using Fisher's exact test and the Bonferroni-Hochberg correction to identify differentially spliced exons associated with autism. Differential splicing events were considered significant if they fulfilled both criteria of FDR<0.1 and % inc difference between autism and controls >15%.

GWAS Set Enrichment Analysis.

GWAS enrichment analysis was performed as previously described in ref. 38 with the main modification that we generated the null distribution, using permutation of gene labels rather than permutation of case/control labels, because the raw genotyping data was not available for all data sets. This approach has been proposed as an acceptable alternative to phenotype label permutation (38) and has been previously used for set enrichment analyses of GWAS data (39). For all genes that met the robust expression criteria in our data set, we mapped the SNPs present on the ILLUMINA 550 k platform located within the transcript boundaries and an additional 20 kb on the 5′ end and 10 kb on the 3′ end. Each gene was assigned a GWAS significance value consisting of the lowest P value of all SNPs mapped to it. A gene set enrichment score (ES) based on the Kolmogorov-Smirnov statistic was calculated as previously described (38) using the 2 log(P-value). The null distribution was generated by 10,000 random permutations of gene labels in the list of genes/P-value pairs and an enrichment score ESp was calculated for each permutation. To correct for the gene set size, the enrichment scores were scaled by subtracting the mean and dividing by the standard deviation of ESp. The resulting z-scores were used to calculate the significance p value.

Primers for q-RT PCR validation of microarray data Gene F/R Sequence (5′-3′) CADPS2 F TACCCCTTCAACGCCAAG (SEQ ID NO: 45) CADPS2 R CCTGGAACCGTTCTTTCAGT (SEQ ID NO: 46) CAMK1G F CTGTAGGAGCTGGAGTGGGA (SEQ ID NO: 47) CAMK1G R TTCTTCCTTTCGACCCATTG (SEQ ID NO: 48) CD44 F GACAAGTTTTGGTGGCACG (SEQ ID NO: 49) CD44 R CACGTGGAATACACCTGCAA (SEQ ID NO: 50) CDKN1A F ACCGAGGCACTCAGAGGAG (SEQ ID NO: 51) CDKN1A R GCCATTAGCGCATCACAGT (SEQ ID NO: 52) GADD45B F ACAGTGGGGGTGTACGAGTC (SEQ ID NO: 53) GADD45B R GATGTCATCCTCCTCCTCCTC (SEQ ID NO: 54) HAPLN4 F AATGAGCTGGAAGATGACGC (SEQ ID NO: 55) HAPLN4 R GAAGGTCAGCTTGTATCGGC (SEQ ID NO: 56) IFITM3 F ATGTCGTCTGGTCCCTGTTC (SEQ ID NO: 57) IFITM3 R CCAACCATCTTCCTGTCCC (SEQ ID NO: 58) NEFH F CAGGACCTGCTCAATGTCAA (SEQ ID NO: 59) NEFH R CAAAGCCAATCCGACACTCT (SEQ ID NO: 60) SERPINA3 F GGAACTAGGGGGAAAATCACA (SEQ ID NO: 61) SERPINA3 R GTCAAAGGGCATCTCCCAT (SEQ ID NO: 62) STAT4 F GGTCGTGTTTCCAAAGAGAAA (SEQ ID NO: 63) STAT4 R TGCAGCCAATATTCTCCTCTC (SEQ ID NO: 64) VAMP1 F CAGCCTCCGGAGAGGAA (SEQ ID NO: 65) VAMP1 R CAGTCCCTTCTGTCCCTTCA (SEQ ID NO: 66) Primers for semi-quantitative RT PCR validation of differential splicing events Gene Inclusion Exclusion F/R Sequence (5′-3′) event (bp) event (bp) AGFG1 F TCAGACCAATGCCAGAGGAGC (SEQ ID NO: 67) 213 165 AGFG1 R GCTGTCTGTTGAGGGAAAGCTG (SEQ ID NO: 68) 213 165 CDC42BPA F GTCCTGGAGATGGAATACAGATC (SEQ ID NO: 69) 342 156 CDC42BPA R CTGACAAGCCACTGCTAGCAC (SEQ ID NO: 70) 342 156 EHBP1 F CAGCAAGATGAAGAGCGACGTC (SEQ ID NO: 71) 378 270 EHBP1 R CATGTCCTGCTCTGAGCTCTC (SEQ ID NO: 72) 378 270 GRIN1 F AGCATCCACCTGAGCTTCCTG (SEQ ID NO: 73) 341 278 GRIN1 R CTGGCAGAAAGGATGATGACCC (SEQ ID NO: 74) 341 278 NRCAM F TTCCTGCCAACAAGACACGGTG (SEQ ID NO: 75) 223 187 NRCAM R AGACCAATGAACCAGCCCTGAG (SEQ ID NO: 76) 223 187 RPN2 F ATGCTGGGACTCATGTATGTCTAC (SEQ ID NO: 77) 264 216 RPN2 R CTTCTCATACTGTGAATTGTTCTTGAC (SEQ ID NO: 78) 264 216 SORBS1 F GTCGGGATATAAGCCCAGAAGAG (SEQ ID NO: 79) 231 129 SORBS1 R CAGGAGTCTCTGAAGAAATTTCCG (SEQ ID NO: 80) 231 129 F-forward primer, R-reverse primer

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TABLES

TABLE A DIFFERENTIAL EXPRESSION IN DASD GENES Illustrative probes differentially expressed between autism and control samples are listed below. ILLUMINA Probe ID. FC = fold change. Probe ID Gene Symbol Cortex_Autism vs Controls_FC ILMN_2069224 PVALB 0.384281104 ILMN_2165463 HAPLN4 0.450365646 ILMN_1812824 SST 0.487002818 ILMN_1801302 SCN1B 0.503271564 ILMN_1785202 STAT4 0.519700809 ILMN_1705153 NEFH 0.535060416 ILMN_1737611 VAMP1 0.535167674 ILMN_2281786 RTN1 0.548921039 ILMN_1728301 GAD2 0.549928388 ILMN_2292646 GAD1 0.575469672 ILMN_2081813 PCSK1 0.576302013 ILMN_1668035 CRH 0.578603948 ILMN_1697512 SLC32A1 0.580940223 ILMN_1748983 RTN4 0.592773439 ILMN_2330845 NSF 0.608527579 ILMN_2408080 SNAP25 0.610725452 ILMN_1761903 KCNS1 0.613791146 ILMN_1765966 CHGB 0.620605007 ILMN_2413964 GABRG2 0.623626217 ILMN_1757497 VGF 0.625709992 ILMN_2071186 CORT 0.626115552 ILMN_1772627 D4S234E 0.630196216 ILMN_1802633 GABRA1 0.631429459 ILMN_2342554 TAGLN3 0.638467628 ILMN_1672121 LOC387856 0.638807504 ILMN_1779428 LOC387856 0.639435175 ILMN_1684461 CADPS2 0.646152723 ILMN_1674778 ATP6V1G2 0.646571954 ILMN_1788053 SLC25A12 0.646680423 ILMN_2215989 NEFM 0.646766037 ILMN_1806473 NGFRAP1L1 0.649374756 ILMN_1763344 ADCYAP1 0.650123932 ILMN_1789166 SHD 0.65729442 ILMN_1809613 NGEF 0.657683412 ILMN_1704383 TRIM37 0.65772731 ILMN_1713603 PRKCB1 0.658272725 ILMN_1794829 C6orf117 0.65912642 ILMN_2405756 VAMP1 0.659332141 ILMN_1794638 VIP 0.660660313 ILMN_1775566 ATP1A1 0.66137894 ILMN_1652540 C5orf16 0.664060869 ILMN_1731062 NPY 0.667066471 ILMN_1685834 AMPH 0.668795152 ILMN_1758067 RGS4 0.673622398 ILMN_1722559 NEUROD6 0.673944661 ILMN_1735743 FLJ37440 0.676926154 ILMN_1653856 STS-1 0.679758078 ILMN_1718295 STAC2 0.679781142 ILMN_1779241 CRYM 0.679937721 ILMN_1673704 INA 0.680735329 ILMN_2332250 ACOT7 0.681642287 ILMN_1716019 RHBDL3 0.681797213 ILMN_1707137 LOC400566 0.682741904 ILMN_1716988 OPN3 0.686320687 ILMN_1779343 SNCB 0.687477404 ILMN_1774604 PNKD 0.687489982 ILMN_2120210 DSCR1L1 0.687632103 ILMN_2165354 DCAMKL1 0.68996275 ILMN_1672094 DLX1 0.691470434 ILMN_1659820 PTD004 0.692654635 ILMN_1809101 STEAP2 0.694583493 ILMN_2080080 MAP7D2 0.695165716 ILMN_1770653 MAL2 0.695318144 ILMN_1662419 COX7A1 0.695872309 ILMN_1804339 CAMK1G 0.696983027 ILMN_2255579 RAB37 0.69739304 ILMN_1745817 NELL1 0.697418975 ILMN_1809947 FLJ30834 0.697789248 ILMN_1764780 SVOP 0.699929938 ILMN_1756701 MGC4172 0.700484179 ILMN_1788538 NCALD 0.700549331 ILMN_1754727 GPRASP2 0.701019441 ILMN_1801703 CPLX1 0.701679843 ILMN_1810604 ELMOD1 0.703235976 ILMN_1731783 ATP1A1 0.703441219 ILMN_1726666 GPX3 0.703449485 ILMN_1679580 KCNIP4 0.703521823 ILMN_1682326 PCP4 0.704164246 ILMN_1685496 RGS7 0.704244753 ILMN_1664071 TNNT2 0.705182859 ILMN_1682799 STAMBPL1 0.705814226 ILMN_1677439 GLS2 0.706002692 ILMN_1721605 SMYD2 0.706030377 ILMN_1803818 NMNAT2 0.706103782 ILMN_1774350 MYOZ3 0.709551452 ILMN_1767365 PAK1 0.710736634 ILMN_1658094 ZNF365 0.712214367 ILMN_2180371 C12orf24 0.713178503 ILMN_2384409 TAC1 0.714342911 ILMN_1716563 PRKCB1 0.714465631 ILMN_1796069 CBLN2 0.714645886 ILMN_1779264 DSCR2 0.71492472 ILMN_1684040 C6orf190 0.715113325 ILMN_1717799 PRKCE 0.715200483 ILMN_2365569 ICA1 0.715348345 ILMN_1729075 PTHR2 0.71548016 ILMN_2414878 STXBP1 0.715845667 ILMN_1677746 CABP1 0.716591822 ILMN_1751689 CHRM1 0.717655538 ILMN_1797950 EXTL2 0.718804678 ILMN_1793006 TAC3 0.719187578 ILMN_1690397 DYNC1I1 0.719574163 ILMN_1789505 ITPR1 0.720029469 ILMN_1669410 CHGA 0.720484862 ILMN_1654639 HERC6 0.72054581 ILMN_1731612 UCHL5 0.721199072 ILMN_1657855 ACTL6B 0.721203267 ILMN_1658499 SYT13 0.721207061 ILMN_1679796 TOMM20 0.722254827 ILMN_1803036 TARBP1 0.722409415 ILMN_1688188 CADPS 0.722725629 ILMN_1784783 NME5 0.723678541 ILMN_1723837 SLC6A17 0.723939656 ILMN_1715189 LHX6 0.724004197 ILMN_2344298 STEAP2 0.7244329 ILMN_2357542 VIP 0.72454341 ILMN_1699768 CBLN4 0.724750508 ILMN_1808765 ZNF25 0.72506654 ILMN_1656560 DKFZP564O0823 0.72619823 ILMN_2065254 ELAVL4 0.727260104 ILMN_1777261 FAM3C 0.727669273 ILMN_2330966 PTK2B 0.727707779 ILMN_1729165 TCEAL6 0.729243089 ILMN_2381938 ATP2B2 0.729789904 ILMN_1698726 SLC25A27 0.730023038 ILMN_2042792 C13orf16 0.730431658 ILMN_1778371 CCBL2 0.730979547 ILMN_2359168 A2BP1 0.731078044 ILMN_2082209 C20orf100 0.731282208 ILMN_1713803 LOC400566 0.731732794 ILMN_1668411 FHL2 0.731985286 ILMN_1674661 CIRBP 0.732314211 ILMN_2181125 NAPB 0.73350304 ILMN_1800270 GABRG2 0.734294209 ILMN_2345319 PREPL 0.734922956 ILMN_1758825 ABLIM2 0.735175106 ILMN_1711327 TRIM37 0.735739611 ILMN_1651745 TMEM25 0.735822073 ILMN_1699623 FAM81A 0.735837506 ILMN_1786966 ATRNL1 0.735978083 ILMN_1676998 SCN2B 0.736444548 ILMN_1656300 GFRA2 0.73647511 ILMN_1656129 SLC39A10 0.736522521 ILMN_2404256 PTPRT 0.736687579 ILMN_1659285 DSCR2 0.736975199 ILMN_1685690 SMCY 0.737126414 ILMN_1734695 MAP4 0.737278688 ILMN_1669382 CPLX2 0.737419865 ILMN_2210199 NLGN4Y 0.738054357 ILMN_1798841 PLCXD3 0.738823623 ILMN_1741156 ARMCX5 0.739252187 ILMN_1720158 ETS2 0.739433777 ILMN_1773307 NAP1L5 0.739497205 ILMN_2056975 HPRT1 0.739677847 ILMN_1814316 A2BP1 0.739897483 ILMN_1665547 CADPS 0.740132193 ILMN_1694240 MAP2K1 0.740637561 ILMN_1663092 CITED2 0.741797151 ILMN_1671766 F12 0.744289746 ILMN_1659801 ATP6V1C1 0.744325957 ILMN_1690179 CRYM 0.744494171 ILMN_1666904 SLC17A6 0.745337431 ILMN_1805737 PFKP 0.745394034 ILMN_1732066 CKMT1A 0.745683317 ILMN_1705066 BTBD11 0.745921455 ILMN_1730794 SERTAD4 0.746354594 ILMN_1751569 C1orf173 0.746375475 ILMN_1795826 ATP6V0D1 0.746760234 ILMN_2124241 MUM1L1 0.74676485 ILMN_1728747 STXBP1 0.746770797 ILMN_2288784 CCDC34 0.74710188 ILMN_1754076 CACNA2D3 0.747283189 ILMN_2171295 PFTK1 0.7478083 ILMN_1652244 POPDC3 0.747964426 ILMN_1731507 A2BP1 0.748209685 ILMN_2400292 MAPK9 0.748997044 ILMN_1697418 RBM9 0.749556808 ILMN_2368773 FAM3C 0.749756884 ILMN_1807177 KIAA1797 0.749838686 ILMN_1781151 ARMC8 0.749960486 ILMN_1814787 ICA1 0.750939994 ILMN_2044061 OR2L13 0.751897754 ILMN_2075643 ANKRD29 0.752573009 ILMN_1765001 NAT6 0.752975424 ILMN_1669631 GLRB 0.753238813 ILMN_1659086 NEFL 0.753285609 ILMN_2229379 KIT 0.753832901 ILMN_1736015 PHF17 0.75409227 ILMN_1658071 ATP1B1 0.754161622 ILMN_1757387 UCHL1 0.754638858 ILMN_1771652 BAIAP2L2 0.755023826 ILMN_1694799 PIAS2 0.755044426 ILMN_1788886 TOX 0.755227223 ILMN_2415073 ATP2B3 0.755531037 ILMN_1750181 TESC 0.755901508 ILMN_1666625 KIF17 0.756350784 ILMN_1695947 SCN4B 0.756478639 ILMN_1793615 ME3 0.756690571 ILMN_1715384 B3GNT6 0.758204908 ILMN_1803045 TUBGCP5 0.758376695 ILMN_1702296 CART 0.758584037 ILMN_2223805 TSGA14 0.758646088 ILMN_1723971 SLC29A1 0.758867247 ILMN_1688452 LCMT1 0.75918473 ILMN_1695003 PCSK2 0.759381713 ILMN_1726743 MRPS30 0.759483809 ILMN_1719998 C9orf45 0.759592351 ILMN_2087941 ENTPD3 0.759680776 ILMN_2218780 PPM2C 0.759883578 ILMN_2333319 PTBP1 1.300132976 ILMN_1656368 ALDH4A1 1.300608591 ILMN_2176037 GNA13 1.3014974 ILMN_1733559 CDC14B 1.303579166 ILMN_1796734 SPARC 1.303906501 ILMN_2376625 VHL 1.30431452 ILMN_1758315 SLC9A9 1.304687298 ILMN_1728049 S100A16 1.306483572 ILMN_2286987 LOC388419 1.307115281 ILMN_1771599 PLOD2 1.307729013 ILMN_1693421 RPN2 1.308725149 ILMN_1789830 CFLAR 1.309526066 ILMN_2255310 RPS15A 1.30991181 ILMN_1815745 SOX4 1.310265138 ILMN_1765446 EMP3 1.310916707 ILMN_1786347 TNPO1 1.312730027 ILMN_1728426 INPPL1 1.31277277 ILMN_1774602 FBLN2 1.313140283 ILMN_1702487 SGK 1.313405396 ILMN_1736178 AEBP1 1.315796307 ILMN_1682139 RAI14 1.316216905 ILMN_1703946 ADORA2B 1.316279075 ILMN_1727402 HCLS1 1.316576577 ILMN_1714335 RDH10 1.316706415 ILMN_1773389 PLTP 1.316991139 ILMN_1690621 MASS1 1.317088722 ILMN_1654966 SCARA3 1.317149318 ILMN_1684391 PLOD1 1.317897837 ILMN_1753342 SAT 1.319471258 ILMN_1740466 FAM46A 1.31987569 ILMN_1695290 PLEKHC1 1.320032643 ILMN_1668374 ITGB5 1.320222486 ILMN_2177156 SOX2 1.320334654 ILMN_1805466 SOX9 1.320689194 ILMN_1682717 IER3 1.323513955 ILMN_1752075 MYBPC1 1.324220106 ILMN_1668345 OAF 1.325429692 ILMN_2176592 BCHE 1.325841151 ILMN_2188521 PVRL3 1.325901636 ILMN_1687301 CSPG2 1.327319843 ILMN_1660125 SFMBT2 1.327689835 ILMN_1738854 CACHD1 1.32771151 ILMN_1682034 HEY2 1.327996031 ILMN_1702231 C1orf54 1.328534332 ILMN_1781400 SLC7A2 1.328570512 ILMN_1761247 PIR 1.328867166 ILMN_2259119 PRMT2 1.329225118 ILMN_1681679 TSPO 1.329660203 ILMN_1803788 LGALS3 1.3298149 ILMN_1719599 SYTL4 1.329919797 ILMN_1675268 LRP4 1.330020664 ILMN_1736567 CD74 1.331116886 ILMN_2130441 HLA-H 1.332048268 ILMN_1683133 KLF15 1.332471322 ILMN_1777397 MSX1 1.333305969 ILMN_2197365 RGS2 1.334884967 ILMN_1654289 ELK1 1.334923332 ILMN_2179717 C9orf61 1.336291611 ILMN_1768973 HIST2H2AC 1.336334149 ILMN_1774836 PLOD3 1.337460354 ILMN_1796749 KIF1C 1.33827213 ILMN_1701603 ALPL 1.338442458 ILMN_2184966 ZHX2 1.338828124 ILMN_1753182 COL20A1 1.339243854 ILMN_1795865 FGFRL1 1.339610467 ILMN_1731418 SP110 1.340284786 ILMN_1782439 CNN3 1.340935205 ILMN_1802167 ALDH1L1 1.34107932 ILMN_1709479 YAP1 1.341768801 ILMN_1757338 PLSCR4 1.341989739 ILMN_1698934 CMTM7 1.342479655 ILMN_1804448 MSI2 1.342625697 ILMN_1767523 IL17RB 1.343641644 ILMN_1680110 C10orf116 1.34426038 ILMN_1732154 BCAN 1.344655146 ILMN_2203950 HLA-A 1.345111215 ILMN_2085862 SLC15A3 1.3459625 ILMN_1728197 CLDN5 1.346396612 ILMN_2200659 SNHG5 1.346663975 ILMN_2155719 NBPF10 1.348762329 ILMN_2056032 CD99 1.35087478 ILMN_1798360 CMKOR1 1.351860155 ILMN_2144426 HIST2H2AA3 1.352184014 ILMN_1736670 PPP1R3C 1.353817566 ILMN_2216157 GNA12 1.354245467 ILMN_1749213 SDF2L1 1.354720976 ILMN_1781952 MGST1 1.354736305 ILMN_2058251 VIM 1.354806838 ILMN_1668863 LYPD1 1.355006983 ILMN_1761280 NTSR2 1.360418039 ILMN_1744897 KCNN3 1.36042729 ILMN_2390853 CTSH 1.36272381 ILMN_1723358 SCARA3 1.362824308 ILMN_1675848 MRCL3 1.363338259 ILMN_1773427 ANKRD15 1.363898988 ILMN_1661755 C9orf88 1.363944079 ILMN_2124769 YBX1 1.36765071 ILMN_1705247 ACSL5 1.367657046 ILMN_1723035 OLR1 1.36769625 ILMN_1670638 PITPNC1 1.367892968 ILMN_1673769 KCNG1 1.370111644 ILMN_1750386 NPPA 1.370563137 ILMN_1670379 ANTXR1 1.371195234 ILMN_1747683 AQP4 1.371277292 ILMN_1693826 HAVCR2 1.371614522 ILMN_1753312 PLXDC2 1.374163389 ILMN_1773576 CPNE3 1.375129641 ILMN_1712583 METRN 1.3780468 ILMN_1732410 SLC16A9 1.380328943 ILMN_1660436 HSPA1B 1.381550236 ILMN_2184184 ANXA1 1.381923015 ILMN_1676616 PTPRZ1 1.382212794 ILMN_1751079 TAP1 1.385550053 ILMN_1809477 CARHSP1 1.385652772 ILMN_1680132 IGSF4 1.386411325 ILMN_2205963 C10orf54 1.386894281 ILMN_2352097 GPR56 1.386938495 ILMN_2384122 GPR56 1.387425194 ILMN_2385220 DFFA 1.388729352 ILMN_2128750 PTTG1IP 1.390959596 ILMN_1701025 EPHX1 1.391766624 ILMN_2227011 ACSBG1 1.394607348 ILMN_1812262 DDR1 1.396131103 ILMN_1775170 MT1X 1.398211578 ILMN_1693334 P4HA1 1.399932983 ILMN_1744604 CYBA 1.401760599 ILMN_1685194 CLDN10 1.402888402 ILMN_1803988 MCL1 1.403682662 ILMN_1653028 COL4A1 1.404711523 ILMN_1668039 GYPC 1.404728614 ILMN_2138589 MERTK 1.405217169 ILMN_1760027 WAS 1.407637733 ILMN_1670535 NDRG2 1.411625495 ILMN_1709747 ENDOGL1 1.417857251 ILMN_1747019 PDYN 1.419964487 ILMN_1724533 LY96 1.422076541 ILMN_2302118 CCDC50 1.422142687 ILMN_1757406 HIST1H1C 1.422752303 ILMN_1732296 ID3 1.423123586 ILMN_1719695 NFKBIZ 1.423475111 ILMN_2109708 ECGF1 1.425340435 ILMN_1705213 TMBIM1 1.426750314 ILMN_2390919 FBLN2 1.427232232 ILMN_1651496 HIST1H2BD 1.427546661 ILMN_1697448 TXNIP 1.429543296 ILMN_2175912 ITGB2 1.431048056 ILMN_1772359 LAPTM5 1.434751146 ILMN_1813741 KCNJ16 1.434771222 ILMN_2190084 VAMP8 1.434805821 ILMN_1813639 C20orf58 1.436080804 ILMN_1778977 TYROBP 1.43820372 ILMN_2336781 SOD2 1.439605517 ILMN_2322996 EYA2 1.442796588 ILMN_1699856 RALGDS 1.445595952 ILMN_2347068 MKNK2 1.445680924 ILMN_1687440 HIPK2 1.446628634 ILMN_1687384 G1P3 1.448875551 ILMN_1806023 JUN 1.451374405 ILMN_2086470 PDGFRA 1.453070176 ILMN_2132982 IGFBP5 1.458937944 ILMN_1740938 APOE 1.459970407 ILMN_2184064 ARRDC4 1.460135156 ILMN_1801246 IFITM1 1.460372479 ILMN_2062468 IGFBP7 1.460457711 ILMN_1685005 TNFRSF1A 1.463416696 ILMN_1671337 SLC2A5 1.46404106 ILMN_1766054 ABCA1 1.477720314 ILMN_2172174 NP 1.486822375 ILMN_2364022 SLC16A3 1.487026141 ILMN_2394777 DTNA 1.489346211 ILMN_1794017 SERTAD1 1.489616024 ILMN_1732071 HIST2H2BE 1.489840157 ILMN_1691717 RHBDF2 1.492220423 ILMN_2138765 ADFP 1.494781928 ILMN_1723480 BST2 1.496075322 ILMN_1675448 ZFP36L1 1.497557616 ILMN_1659895 MSN 1.501647858 ILMN_2090105 TAGLN2 1.502315072 ILMN_1686664 MT2A 1.507462376 ILMN_1713124 AKR1C3 1.509948155 ILMN_1718977 GADD45B 1.51425696 ILMN_2074860 RN7SK 1.516139975 ILMN_1698732 PALLD 1.518206478 ILMN_2361603 NDRG2 1.518558116 ILMN_2115490 NBPF20 1.52846846 ILMN_1705750 TGM2 1.537833644 ILMN_1701613 RARRES3 1.538608409 ILMN_1652549 DTNA 1.542206535 ILMN_2355168 MGST1 1.543990602 ILMN_1781155 LYN 1.547240495 ILMN_1740015 CD14 1.548489789 ILMN_2169152 SRGN 1.551518608 ILMN_2409167 ANXA2 1.55748268 ILMN_1662932 LCP1 1.565422786 ILMN_1728478 CXCL16 1.566911305 ILMN_1697176 GFAP 1.573654136 ILMN_2165753 LOC649853 1.589360981 ILMN_1707727 ANGPTL4 1.591563923 ILMN_1770338 TM4SF1 1.595979975 ILMN_1651498 GADD45G 1.59911241 ILMN_1661599 DDIT4 1.611960674 ILMN_1753143 RHPN2 1.620718361 ILMN_1729188 HAMP 1.623494622 ILMN_1801205 GPNMB 1.626693272 ILMN_1684982 PDK4 1.627757229 ILMN_1775304 DNAJB1 1.632662357 ILMN_2046730 S100A10 1.63345915 ILMN_1730201 DTNA 1.636094723 ILMN_1805750 IFITM3 1.675939595 ILMN_1756982 CLIC1 1.682842296 ILMN_1673352 IFITM2 1.682897581 ILMN_2054297 PTGS2 1.688191961 ILMN_1700183 AGTRL1 1.692387336 ILMN_1789074 HSPA1A 1.697058402 ILMN_1803429 CD44 1.700522592 ILMN_1774077 GBP2 1.712235274 ILMN_1782050 CEBPD 1.747427977 ILMN_1720829 ZFP36 1.754441848 ILMN_1785902 C1QC 1.75467867 ILMN_2302757 FCGBP 1.770651662 ILMN_1674236 HSPB1 1.779867804 ILMN_1711566 TIMP1 1.781871241 ILMN_1796409 C1QB 1.791729263 ILMN_1735502 MGC33846 1.800927745 ILMN_2396444 CD14 1.830377645 ILMN_1789007 APOC1 1.839643827 ILMN_1708934 ADM 1.856402442 ILMN_1782788 CSDA 1.874264646 ILMN_1720282 NQO1 1.875783057 ILMN_1801616 EMP1 1.955142358 ILMN_1767556 C10orf10 1.998536768 ILMN_1784602 CDKN1A 2.12220785 ILMN_1659766 BAG3 2.177420749 ILMN_1729801 S100A8 2.32258342 ILMN_1788874 SERPINA3 2.745885705

TABLE B  ILLUSTRATIVE HUMAN DASD POLYNUCLEOTIDE SEQUENCES INDENTIFIED USING THE DISCLOSURE PRESENTED HEREIN ACOT7 Entrez ID: 11332; OMIM: 602587; Uniprot ID: BACH_HUMAN; ENSEMBL ID: ENSG00000097021 ATGGCGCGGCCCGGGCTCATTCATTCCGCGCCGGGCCTGCCAGACACCTGCGCCCTTCTGCAGCCGCCC GCCGCATCCGCCGCCGCAGCCCCCAGCATGTCGGGCCCAGACGTCGAGACGCCGTCCGCCATCCAGATC TGCCGGATCATGCGGCCAGATGATGCCAACGTGGCCGGCAATGTCCACGGGGGGACCATCCTGAAGATG ATCGAGGAGGCAGGCGCCATCATCAGCACCCGGCATTGCAACAGCCAGAACGGGGAGCGCTGTGTGGCC GCCCTGGCTCGTGTCGAGCGCACCGACTTCCTGTCTCCCATGTGCATCGGTGAGGTGGCGCATGTCAGC GCGGAGATCACCTACACCTCCAAGCACTCTGTGGAGGTGCAGGTCAACGTGATGTCCGAAAACATCCTC ACAGGTGCCAAAAAGCTGACCAATAAGGCCACCCTGTGGTATGTGCCCCTGTCGCTGAAGAATGTGGAC AAGGTCCTCGAGGTGCCTCCTGTTGTGTATTCCCGGCAGGAGCAGGAGGAGGAGGGCCGGAAGCGGTAT GAAGCCCAGAAGCTGGAGCGCATGGAGACCAAGTGGAGGAACGGGGACATCGTCCAGCCAGTCCTCAAC CCAGAGCCGAACACTGTCAGCTACAGCCAGTCCAGCTTGATCCACCTGGTGGGGCCTTCAGACTGCACC CTGCACGGCTTTGTGCACGGAGGTGTGACCATGAAGCTCATGGATGAGGTCGCCGGGATCGTGGCTGCA CGCCACTGCAAGACCAACATCGTCACAGCTTCCGTGGACGCCATTAATTTTCATGACAAGATCAGAAAA GGCTGCGTCATCACCATCTCGGGACGCATGACCTTCACGAGCAATAAGTCCATGGAGATCGAGGTGTTG GTGGACGCCGACCCTGTTGTGGACAGCTCTCAGAAGCGCTACCGGGCCGCCAGTGCCTTCTTCACCTAC GTGTCGCTGAGCCAGGAAGGCAGGTCGCTGCCTGTGCCCCAGCTGGTGCCCGAGACCGAGGACGAGAAG AAGCGCTTTGAGGAAGGCAAAGGGCGGTACCTGCAGATGAAGGCGAAGCGACAGGGCCACGCGGAGCCT CAGCCCTAG (SEQ ID NO: 1) ALDH4A1 Entrez ID: 8659; OMIM: 606811; Uniprot ID: AL4A1_HUMAN; ENSEMBL ID: ENSG00000159423 ATGGAAGCCATCCCATGCGTGGTGGGGGATGAGGAGGTGTGGACGTCGGACGTGCAGTACCAAGTGTCG CCTTTTAACCATGGACATAAGGTGGCCAAGTTCTGTTATGCAGACAAGAGCCTGCTCAACAAAGCCATT GAGGCTGCCCTGGCTGCCCGGAAAGAGTGGGACCTGAAGCCTATTGCAGACCGGGCCCAGATCTTCCTG AAGGCGGCAGACATGCTGAGTGGGCCGCGCAGGGCTGAGATCCTCGCCAAGACCATGGTGGGACAGGGT AAGACCGTGATCCAAGCGGAGATTGACGCTGCAGCGGAACTCATCGACTTCTTCCGGTTCAATGCCAAG TATGCGGTGGAGCTGGAGGGGCAGCAGCCCATCAGCGTGCCCCCGAGCACCAACAGCACGGTGTACCGG GGTCTGGAGGGCTTCGTGGCGGCCATCTCGCCCTTTAACTTCACTGCAATCGGCGGCAACCTGGCGGGG GCACCGGCCCTGATGGGCAACGTGGTCCTATGGAAGCCCAGTGACACTGCCATGCTGGCCAGCTATGCT GTCTACCGCATCCTTCGGGAGGCTGGCCTGCCCCCCAACATCATCCAGTTTGTGCCAGCTGATGGGCCC CTATTTGGGGACACTGTCACCAGCTCAGAGCACCTCTGTGGCATCAACTTCACAGGCAGTGTGCCCACC TTCAAACACCTGTGGAAGCAGGTGGCCCAGAACCTGGACCGGTTCCACACCTTCCCACGCCTGGCTGGA GAGTGCGGCGGAAAGAACTTCCACTTCGTGCACCGCTCGGCCGACGTGGAGAGCGTGGTGAGCGGGACC CTCCGCTCAGCCTTCGAGTACGGTGGCCAGAAGTGTTCCGCGTGCTCGCGTCTCTACGTGCCGCACTCG CTGTGGCCGCAGATCAAAGGGCGGCTGCTGGAGGAGCACAGTCGGATCAAAGTGGGCGACCCTGCAGAG GATTTTGGGACCTTCTTCTCTGCAGTGATTGATGCCAAGTCCTTTGCCCGTATCAAGAAGTGGCTGGAG CACGCACGCTCCTCACCCAGCCTCACCATCCTGGCCGGGGGCAAGTGTGATGACTCCGTGGGCTACTTT GTGGAGCCCTGCATCGTGGAGAGCAAGGACCCTCAGGAGCCCATCATGAAGGAGGAGATCTTCGGGCCT GTACTGTCTGTGTACGTCTACCCGGATGACAAGTACAAGGAGACGCTGCAGCTGGTTGACAGCACCACC AGCTATGGCCTCACGGGGGCAGTGTTCTCCCAGGATAAGGACGTCGTGCAGGAGGCCACAAAGGTGCTG AGGAATGCTGCCGGCAACTTCTACATCAACGACAAGTCCACTGGCTCGATAGTGGGCCAGCAGCCCTTT GGGGGGGCCCGAGCCTCTGGAACCAATGACAAGCCAGGGGGCCCACACTACATCCTGCGCTGGACGTCG CCGCAGGTCATCAAGGAGACACATAAGCCCCTGGGGGACTGGAGCTACGCGTACATGCAGTGA (SEQ ID NO: 2) ATP1B1 Entrez ID: 8659; OMIM: 606811; Uniprot ID: AL4A1_HUMAN; ENSEMBL ID: ENSG00000159423 ATGGCCCGCGGGAAAGCCAAGGAGGAGGGCAGCTGGAAGAAATTCATCTGGAACTCAGAGAAGAAGGAG TTTCTGGGCAGGACCGGTGGCAGTTGGTTTAAGATCCTTCTATTCTACGTAATATTTTATGGCTGCCTG GCTGGCATCTTCATCGGAACCATCCAAGTGATGCTGCTCACCATCAGTGAATTTAAGCCCACATATCAG GACCGAGTGGCCCCGCCAGGATTAACACAGATTCCTCAGATCCAGAAGACTGAAATTTCCTTTCGTCCT AATGATCCCAAGAGCTATGAGGCATATGTACTGAACATAGTTAGGTTCCTGGAAAAGTACAAAGATTCA GCCCAGAGGGATGACATGATTTTTGAAGATTGTGGCGATGTGCCCAGTGAACCGAAAGAACGAGGAGAC TTTAATCATGAACGAGGAGAGCGAAAGGTCTGCAGATTCAAGCTTGAATGGCTGGGAAATTGCTCTGGA TTAAATGATGAAACTTATGGCTACAAAGAGGGCAAACCGTGCATTATTATAAAGCTCAACCGAGTTCTA GGCTTCAAACCTAAGCCTCCCAAGAATGAGTCCTTGGAGACTTACCCAGTGATGAAGTATAACCCAAAT GTCCTTCCCGTTCAGTGCACTGGCAAGCGAGATGAAGATAAGGATAAAGTTGGAAATGTGGAGTATTTT GGACTGGGCAACTCCCCTGGTTTTCCTCTGCAGTATTATCCGTACTATGGCAAACTCCTGCAGCCCAAA TACCTGCAGCCCCTGCTGGCCGTACAGTTCACCAATCTTACCATGGACACTGAAATTCGCATAGAGTGT AAGGCGTACGGTGAGAACATTGGGTACAGTGAGAAAGACCGTTTTCAGGGACGTTTTGATGTAAAAATT GAAGTTAAGAGCTGA (SEQ ID NO: 3) ATP6V0D1 Entrez ID: 9114; OMIM: 607028; Uniprot ID: VA0D1_HUMAN; ENSEMBL ID: ENSG00000159720 ATGTCGTTCTTCCCGGAGCTTTACTTTAACGTGGACAATGGCTACTTGGAGGGACTGGTGCGCGGCCTG AAGGCCGGGGTGCTCAGCCAGGCCGACTACCTCAACCTGGTGCAGTGCGAGACGCTAGAGGACTTGAAA CTGCATCTGCAGAGCACTGATTATGGTAACTTCCTGGCCAACGAGGCATCACCTCTGACGGTGTCAGTC ATCGATGACCGGCTCAAGGAGAAGATGGTGGTGGAGTTCCGCCACATGAGGAACCATGCCTATGAGCCA CTCGCCAGCTTCCTAGACTTCATTACTTACAGTTACATGATCGACAACGTGATCCTGCTCATCACAGGC ACGCTGCACCAGCGCTCCATCGCTGAGCTCGTGCCCAAGTGCCACCCACTAGGCAGCTTCGAGCAGATG GAGGCCGTGAACATTGCTCAGACACCTGCTGAGCTCTACAATGCCATTCTGGTGGACACGCCTCTTGCG GCTTTTTTCCAGGACTGCATTTCAGAGCAGGACCTTGACGAGATGAACATCGAGATCATCCGCAACACC CTCTACAAGGCCTACCTGGAGTCCTTCTACAAGTTCTGCACCCTACTGGGCGGGACTACGGCTGATGCC ATGTGCCCCATCCTGGAGTTTGAAGCAGACCGCCGCGCCTTCATCATCACCATCAATTCTTTCGGCACA GAGCTGTCCAAAGAGGACCGTGCCAAGCTCTTTCCACACTGTGGGCGGCTCTACCCTGAGGGCCTGGCG CAGCTGGCTCGGGCTGACGACTATGAACAGGTCAAGAACGTGGCCGATTACTACCCGGAGTACAAGCTG CTCTTCGAGGGTGCAGGTAGCAACCCTGGAGACAAGACGCTGGAGGACCGATTCTTTGAGCACGAGGTA AAGCTGAACAAGTTGGCCTTCCTGAACCAGTTCCACTTTGGTGTCTTCTATGCCTTCGTGAAGCTCAAG GAGCAGGAGTGTCGCAACATCGTGTGGATCGCTGAATGTATCGCCCAGCGCCACCGCGCCAAAATCGAC AACTACATCCCTATCTTCTAG (SEQ ID NO: 4) C1orf54 Entrez ID: 79630; OMIM:; Uniprot ID: CA054_HUMAN; ENSEMBL ID:  ENSG00000118292 ATGGATGTCCTCTTTGTAGCCATCTTTGCTGTGCCACTTATCCTGGGACAAGAATATGAGGATGAAGAA AGACTGGGAGAGGATGAATATTATCAGGTGGTCTATTATTATACAGTCACCCCCAGTTATGATGACTTT AGTGCAGATTTCACCATTGATTACTCCATATTTGAGTCAGAGGACAGGCTGAACAGGTTGGATAAGGAC ATAACAGAAGCAATAGAGACTACCATTAGTCTTGAAACAGCACGTGCAGACCATCCGAAGCCTGTAACT GTGAAACCAGTAACAACGGAACCTAGTCCAGATCTGAACGATGCCGTGTCCAGTTTGCGAAGTCCTATT CCCCTCCTCCTGTCGTGTGCCTTTGTTCAGGTGGGGATGTATTTCATGTAG (SEQ ID NO: 5) CD74 Entrez ID: 972; OMIM: 142790; Uniprot ID: HG2A_HUMAN; ENSEMBL ID:  ENSG00000019582 ATGCACAGGAGGAGAAGCAGGAGCTGTCGGGAAGATCAGAAGCCAGTCATGGATGACCAGCGCGACCTT ATCTCCAACAATGAGCAACTGCCCATGCTGGGCCGGCGCCCTGGGGCCCCGGAGAGCAAGTGCAGCCGC GGAGCCCTGTACACAGGCTTTTCCATCCTGGTGACTCTGCTCCTCGCTGGCCAGGCCACCACCGCCTAC TTCCTGTACCAGCAGCAGGGCCGGCTGGACAAACTGACAGTCACCTCCCAGAACCTGCAGCTGGAGAAC CTGCGCATGAAGCTTCCCAAGCCTCCCAAGCCTGTGAGCAAGATGCGCATGGCCACCCCGCTGCTGATG CAGGCGCTGCCCATGGGAGCCCTGCCCCAGGGGCCCATGCAGAATGCCACCAAGTATGGCAACATGACA GAGGACCATGTGATGCACCTGCTCCAGAGTCACTGGAACTGGAGGACCCGTCTTCTGGGCTGGGTGTGA (SEQ ID NO: 6) CEBPD Entrez ID: 1052; OMIM: 116898; Uniprot ID: CEBPD_HUMAN; ENSEMBL ID: ENSG000002218693 ATGAGCGCCGCGCTCTTCAGCCTGGACGGCCCGGCGCGCGGCGCGCCCTGGCCTGCGGAGCCTGCGCCC TTCTACGAACCGGGCCGGGCGGGCAAGCCGGGCCGCGGGGCCGAGCCAGGGGCCCTAGGCGAGCCAGGC GCCGCCGCCCCCGCCATGTACGACGACGAGAGCGCCATCGACTTCAGCGCCTACATCGACTCCATGGCC GCCGTGCCCACCCTGGAGCTGTGCCACGACGAGCTCTTCGCCGACCTCTTCAACAGCAATCACAAGGCG GGCGGCGCGGGGCCCCTGGAGCTTCTTCCCGGCGGCCCCGCGCGCCCCTTGGGCCCGGGCCCTGCCGCT CCCCGCCTGCTCAAGCGCGAGCCCGACTGGGGCGACGGCGACGCGCCCGGCTCGCTGTTGCCCGCGCAG GTGGCCGCGTGCGCACAGACCGTGGTGAGCTTGGCGGCCGCAGGGCAGCCCACCCCGCCCACGTCGCCG GAGCCGCCGCGCAGCAGCCCCAGGCAGACCCCCGCGCCCGGCCCCGCCCGGGAGAAGAGCGCCGGCAAG AGGGGCCCGGACCGCGGCAGCCCCGAGTACCGGCAGCGGCGCGAGCGCAACAACATCGCCGTGCGCAAG AGCCGCGACAAGGCCAAGCGGCGCAACCAGGAGATGCAGCAGAAGTTGGTGGAGCTGTCGGCTGAGAAC GAGAAGCTGCACCAGCGCGTGGAGCAGCTCACGCGGGACCTGGCCGGCCTCCGGCAGTTCTTCAAGCAG CTGCCCAGCCCGCCCTTCCTGCCGGCCGCCGGGACAGCAGACTGCCGGTAA (SEQ ID NO: 7) CFLAR Entrez ID: 8837; OMIM: 603599; Uniprot ID: CFLAR_HUMAN; ENSEMBL ID: ENSG00000003402 ATGTCTGCTGAAGTCATCCATCAGGTTGAAGAAGCACTTGATACAGATGAGAAGGAGATGCTGCTCTTT TTGTGCCGGGATGTTGCTATAGATGTGGTTCCACCTAATGTCAGGGACCTTCTGGATATTTTACGGGAA AGAGGTAAGCTGTCTGTCGGGGACTTGGCTGAACTGCTCTACAGAGTGAGGCGATTTGACCTGCTCAAA CGTATCTTGAAGATGGACAGAAAAGCTGTGGAGACCCACCTGCTCAGGAACCCTCACCTTGTTTCGGAC TATAGAGTGCTGATGGCAGAGATTGGTGAGGATTTGGATAAATCTGATGTGTCCTCATTAATTTTCCTC ATGAAGGATTACATGGGCCGAGGCAAGATAAGCAAGGAGAAGAGTTTCTTGGACCTTGTGGTTGAGTTG GAGAAACTAAATCTGGTTGCCCCAGATCAACTGGATTTATTAGAAAAATGCCTAAAGAACATCCACAGA ATAGACCTGAAGACAAAAATCCAGAAGTACAAGCAGTCTGTTCAAGGAGCAGGGACAAGTTACAGGAAT GTTCTCCAAGCAGCAATCCAAAAGAGTCTCAAGGATCCTTCAAATAACTTCAGGCTCCATAATGGGAGA AGTAAAGAACAAAGACTTAAGGAACAGCTTGGCGCTCAACAAGAACCAGTGAAGAAATCCATTCAGGAA TCAGAAGCTTTTTTGCCTCAGAGCATACCTGAAGAGAGATACAAGATGAAGAGCAAGCCCCTAGGAATC TGCCTGATAATCGATTGCATTGGCAATGAGACAGAGCTTCTTCGAGACACCTTCACTTCCCTGGGCTAT GAAGTCCAGAAATTCTTGCATCTCAGTATGCATGGTATATCCCAGATTCTTGGCCAATTTGCCTGTATG CCCGAGCACCGAGACTACGACAGCTTTGTGTGTGTCCTGGTGAGCCGAGGAGGCTCCCAGAGTGTGTAT GGTGTGGATCAGACTCACTCAGGGCTCCCCCTGCATCACATCAGGAGGATGTTCATGGGAGATTCATGC CCTTATCTAGCAGGGAAGCCAAAGATGTTTTTTATTCAGAACTATGTGGTGTCAGAGGGCCAGCTGGAG GACAGCAGCCTCTTGGAGGTGGATGGGCCAGCGATGAAGAATGTGGAATTCAAGGCTCAGAAGCGAGGG CTGTGCACAGTTCACCGAGAAGCTGACTTCTTCTGGAGCCTGTGTACTGCGGACATGTCCCTGCTGGAG CAGTCTCACAGCTCACCATCCCTGTACCTGCAGTGCCTCTCCCAGAAACTGAGACAAGAAAGAAAACGC CCACTCCTGGATCTTCACATTGAACTCAATGGCTACATGTATGATTGGAACAGCAGAGTTTCTGCCAAG GAGAAATATTATGTCTGGCTGCAGCACACTCTGAGAAAGAAACTTATCCTCTCCTACACATAA (SEQ ID NO: 8) CIRBP ENTREZ ID: 1153; OMIM: 602649; UNIPROT ID: CIRBP_HUMAN; ENSEMBL ID: ENSG00000099622 ATGGCATCAGATGAAGGCAAACTTTTTGTTGGAGGGCTGAGTTTTGACACCAATGAGCAGTCGCTGGAG CAGGTCTTCTCAAAGTACGGACAGATCTCTGAAGTGGTGGTTGTGAAAGACAGGGAGACCCAGAGATCT CGGGGATTTGGGTTTGTCACCTTTGAGAACATTGACGACGCTAAGGATGCCATGATGGCCATGAATGGG AAGTCTGTAGATGGACGGCAGATCCGAGTAGACCAGGCAGGCAAGTCGTCAGACAACCGATCCCGTGGG TACCGTGGTGGCTCTGCCGGGGGCCGGGGCTTCTTCCGTGGGGGCCGAGGACGGGGCCGTGGGTTCTCT AGAGGAGGAGGGGACCGAGGCTATGGGGGGAACCGGTTCGAGTCCAGGAGTGGGGGCTACGGAGGCTCC AGAGACTACTATAGCAGCCGGAGTCAGAGTGGTGGCTACAGTGACCGGAGCTCGGGCGGGTCCTACAGA GACAGTTATGACAGTTACGCTACACACAACGAGTAA (SEQ ID NO: 9) CMTM7 Entrez ID: 112616; OMIM: 607890; Uniprot ID: CKLF7_HUMAN; ENSEMBL ID: ENSG00000153551 ATGTCGCACGGAGCCGGGCTCGTCCGCACCACGTGCAGCAGCGGCAGCGCGCTCGGACCCGGGGCCGGC GCGGCCCAGCCCAGCGCGAGCCCCTTGGAGGGGCTGCTGGACCTCAGCTACCCCCGCACCCACGCGGCC CTGCTGAAAGTGGCGCAAATGGTCACCCTGCTGATTGCCTTCATCTGTGTGCGGAGCTCCCTGTGGACC AACTACAGCGCCTACAGCTACTTTGAAGTGGTCACCATTTGCGACTTGATAATGATCCTCGCCTTTTAC CTGGTCCACCTCTTCCGCTTCTACCGCGTGCTCACCTGTATCAGCTGGCCCCTGTCGGAACTTCTGCAC TATTTAATCGGTACCCTGCTCCTCCTCATCGCCTCCATTGTGGCAGCTTCCAAGAGTTACAACCAGAGC GGACTGGTAGCCGGAGCGATCTTTGGTTTCATGGCCACCTTCCTCTGCATGGCAAGCATATGGCTGTCC TATAAGATCTCGTGTGTAACCCAGTCCACAGATGCAGCCGTCTGA (SEQ ID NO: 10) CPNE3 ENTREZ ID: 8895; OMIM: 604207; UNIPROT ID: CPNE3_HUMAN; ENSEMBL ID: ENSG00000085719 ATGGCTGCCCAGTGTGTCACAAAGGTGGCGCTGAATGTTTCCTGTGCCAATCTTTTGGATAAAGATATA GGGTCAAAGTCAGACCCTTTATGTGTGTTGTTTTTGAATACAAGTGGTCAACAGTGGTATGAGGTTGAG CGCACAGAAAGGATTAAGAATTGCTTGAATCCCCAATTTTCCAAGACATTTATTATTGATTACTACTTT GAAGTGGTTCAGAAATTGAAATTTGGGGTTTATGACATCGACAACAAAACTATTGAGCTGAGTGATGAT GACTTCTTAGGGGAATGTGAATGTACCCTTGGACAAATTGTTTCCAGCAAGAAGCTAACTCGACCACTG GTGATGAAAACTGGCAGACCTGCAGGAAAAGGGAGCATTACGATTTCAGCTGAAGAAATAAAAGATAAT AGAGTGGTCTTGTTTGAAATGGAAGCCAGAAAACTGGATAATAAGGATCTATTTGGAAAGTCAGACCCA TACCTGGAATTCCACAAGCAGACATCTGATGGAAACTGGCTAATGGTTCATCGGACAGAGGTTGTTAAA AACAACTTGAATCCTGTTTGGAGGCCTTTCAAGATCTCTCTTAACTCACTGTGTTACGGAGATATGGAC AAAACCATTAAGGTGGAGTGTTATGATTATGACAATGATGGGTCACATGATCTCATTGGAACATTTCAG ACCACCATGACAAAACTGAAAGAAGCCTCCAGAAGCTCACCTGTTGAATTTGAATGCATAAATGAGAAA AAAAGGCAAAAGAAAAAAAGCTACAAGAATTCAGGTGTTATCAGTGTGAAACAGTGTGAGATTACAGTA GAATGCACATTCCTTGACTATATAATGGGAGGATGTCAGCTGAATTTTACTGTGGGAGTGGACTTCACT GGCTCCAATGGTGACCCAAGGTCTCCAGACTCCCTTCATTACATCAGCCCCAATGGCGTTAATGAGTAT TTGACTGCTCTCTGGTCTGTGGGACTGGTCATTCAAGATTATGATGCTGATAAGATGTTTCCAGCTTTT GGTTTTGGCGCTCAGATACCTCCTCAGTGGCAGGTATCACATGAATTTCCAATGAACTTCAACCCATCC AATCCCTACTGCAATGGAATCCAAGGCATTGTAGAGGCGTATCGGTCTTGTCTTCCTCAGATAAAACTC TATGGACCAACTAATTTTTCTCCAATCATAAATCACGTGGCCAGGTTTGCTGCTGCAGCCACGCAACAG CAGACAGCTTCTCAATATTTTGTGCTTTTGATTATTACTGATGGTGTGATCACAGACCTTGATGAAACC AGACAAGCTATAGTTAATGCCTCCAGGCTGCCTATGTCCATCATAATTGTTGGAGTTGGAGGTGCTGAC TTCAGCGCCATGGAGTTTCTGGATGGTGATGGTGGAAGTCTCCGCTCCCCATTGGGCGAAGTGGCCATC AGAGATATTGTCCAGTTTGTGCCTTTCAGACAGTTCCAGAATGCTCCAAAAGAAGCACTTGCTCAGTGT GTCTTGGCAGAGATTCCCCAGCAGGTGGTGGGCTACTTCAATACATACAAACTCCTTCCTCCCAAGAAC CCAGCCACGAAACAACAGAAGCAGTGA (SEQ ID NO: 11) DKFZP564O0823 Entrez ID: 25849; OMIM:; Uniprot ID: PARM1_HUMAN; ENSEMBL ID: ENSG00000169116 ATGGTCTACAAGACTCTCTTCGCTCTTTGCATCTTAACTGCAGGATGGAGGGTACAGAGTCTGCCTACA TCAGCTCCTTTGTCTGTTTCTCTTCCGACAAACATTGTACCACCGACCACCATCTGGACTAGCTCTCCA CAAAACACTGATGCAGACACTGCCTCCCCATCCAACGGCACTCACAACAACTCGGTGCTCCCAGTTACA GCATCAGCCCCAACATCTCTGCTTCCTAAGAACATTTCCATAGAGTCCAGAGAAGAGGAGATCACCAGC CCAGGTTCGAATTGGGAAGGCACAAACACAGACCCCTCACCTTCTGGGTTCTCGTCAACAAGCGGTGGA GTCCACTTAACAACCACGTTGGAGGAACACAGCTCGGGCACTCCTGAAGCAGGCGTGGCAGCTACACTG TCGCAGTCCGCTGCTGAGCCTCCCACACTCATCTCCCCTCAAGCTCCAGCCTCATCACCCTCATCCCTA TCAACCTCACCACCTGAGGTCTTTTCTGCCTCCGTTACTACCAACCATAGCTCCACTGTGACCAGCACC CAACCCACTGGAGCTCCAACTGCACCAGAGTCCCCGACAGAGGAGTCCAGCTCTGACCACACACCCACT TCACATGCCACAGCTGAGCCAGTACCCCAGGAGAAAACACCCCCAACAACTGTGTCAGGCAAAGTGATG TGTGAGCTCATAGACATGGAGACCACCACCACCTTTCCCAGGGTGATCATGCAGGAAGTAGAACATGCA TTAAGTTCAGGCAGCATCGCCGCCATTACCGTGACAGTCATTGCCGTGGTGCTGCTGGTGTTTGGAGTT GCAGCCTACCTAAAAATCAGGCATTCCTCCTATGGAAGACTTTTGGACGACCATGACTACGGGTCCTGG GGAAACTACAACAACCCTCTGTACGATGACTCCTAA (SEQ ID NO: 12) EMP3 Entrez ID: 2014; OMIM: 602335; Uniprot ID: EMP3_HUMAN; ENSEMBL ID:  ENSG00000142227 ATGTCACTCCTCTTGCTGGTGGTCTCAGCCCTTCACATCCTCATTCTTATACTGCTTTTCGTGGCCACT TTGGACAAGTCCTGGTGGACTCTCCCTGGGAAAGAGTCCCTGAATCTCTGGTACGACTGCACGTGGAAC AACGACACCAAAACATGGGCCTGCAGTAATGTCAGCGAGAATGGCTGGCTGAAGGCGGTGCAGGTCCTC ATGGTGCTCTCCCTCATTCTCTGCTGTCTCTCCTTCATCCTGTTCATGTTCCAGCTCTACACCATGCGA CGAGGAGGTCTCTTCTATGCCACCGGCCTCTGCCAGCTTTGCACCAGCGTGGCGGTGTTTACTGGCGCC TTGATCTATGCCATTCACGCCGAGGAGATCCTGGAGAAGCACCCGCGAGGGGGCAGCTTCGGATACTGC TTCGCCCTGGCCTGGGTGGCCTTCCCCCTCGCCCTGGTCAGCGGCATCATCTACATCCACCTACGGAAG CGGGAGTGA (SEQ ID NO: 13) FAM3C Entrez ID: 10447; OMIM: 608618; Uniprot ID: FAM3C_HUMAN; ENSEMBL ID: ENSG00000196937 ATGAGGGTAGCAGGTGCTGCAAAGTTGGTGGTAGCTGTGGCAGTGTTTTTACTGACATTTTATGTTATT TCTCAAGTATTTGAAATAAAAATGGATGCAAGTTTAGGAAATCTATTTGCAAGATCAGCATTGGACACA GCTGCACGTTCTACAAAGCCTCCCAGATATAAGTGTGGGATCTCAAAAGCTTGCCCTGAGAAGCATTTT GCTTTTAAAATGGCAAGTGGAGCAGCCAACGTGGTGGGACCCAAAATCTGCCTGGAAGATAATGTTTTA ATGAGTGGTGTTAAGAATAATGTTGGAAGAGGGATCAATGTTGCCTTGGCAAATGGAAAAACAGGAGAA GTATTAGACACTAAATATTTTGACATGTGGGGAGGAGATGTGGCACCATTTATTGAGTTTCTGAAGGCC ATACAAGATGGAACAATAGTTTTAATGGGAACATACGATGATGGAGCAACCAAACTCAATGATGAGGCA CGGCGGCTCATTGCTGATTTGGGGAGCACATCTATTACTAATCTTGGTTTTAGAGACAACTGGGTCTTC TGTGGTGGGAAGGGCATTAAGACAAAAAGCCCTTTTGAACAGCACATAAAGAACAATAAGGATACAAAC AAATATGAAGGATGGCCTGAAGTTGTAGAAATGGAAGGATGCATCCCCCAGAAGCAAGACTAA (SEQ ID NO: 14) FAM46A Entrez ID: 55603; OMIM: 611357; Uniprot ID: FA46A_HUMAN; ENSEMBL ID: ENSG00000112773 ATGGCGGAGGGTGAAGGGTACTTCGCCATGTCTGAGGACGAGCTGGCCTGCAGCCCCTACATCCCCCTA GGCGGCGACTTCGGCGGCGGCGACTTCGGCGGCGGCGACTTCGGCGGCGGCGACTTCGGCGGTGGCGGC AGCTTCGGTGGGCATTGCTTGGACTATTGCGAAAGCCCTACGGCGCACTGCAATGTGCTGAACTGGGAG CAAGTGCAGCGGCTGGACGGCATCCTGAGCGAGACCATTCCGATTCACGGGCGCGGCAACTTCCCCACG CTCGAGCTGCAGCCGAGCCTGATCGTGAAGGTGGTGCGGCGGCGCCTGGCCGAGAAGCGCATTGGCGTC CGCGACGTGCGCCTCAACGGCTCGGCAGCCAGCCATGTCCTGCACCAGGACAGCGGCCTGGGCTACAAG GACCTGGACCTCATCTTCTGCGCCGACCTGCGCGGGGAAGGGGAGTTTCAGACTGTGAAGGACGTCGTG CTGGACTGCCTGTTGGACTTCTTACCCGAGGGGGTGAACAAAGAGAAGATCACACCACTCACGCTCAAG GAAGCTTATGTGCAGAAAATGGTTAAAGTGTGCAATGACTCTGACCGATGGAGTCTTATATCCCTGTCA AACAACAGTGGCAAAAATGTGGAACTGAAATTTGTGGATTCCCTCCGGAGGCAGTTTGAATTCAGTGTA GATTCTTTTCAAATCAAATTAGACTCTCTTCTGCTCTTTTATGAATGTTCAGAGAACCCAATGACTGAG ACATTTCACCCCACAATAATCGGGGAGAGCGTCTATGGCGATTTCCAGGAAGCCTTTGATCACCTTTGT AACAAGATCATTGCCACCAGGAACCCAGAGGAAATCCGAGGGGGAGGCCTGCTTAAGTACTGCAACCTC TTGGTGAGGGGCTTTAGGCCCGCCTCTGATGAAATCAAGACCCTTCAAAGGTATATGTGTTCCAGGTTT TTCATCGACTTCTCAGACATTGGAGAGCAGCAGAGAAAACTGGAGTCCTATTTGCAGAACCACTTTGTG GGATTGGAAGACCGCAAGTATGAGTATCTCATGACCCTTCATGGAGTGGTAAATGAGAGCACAGTGTGC CTGATGGGACATGAAAGAAGACAGACTTTAAACCTTATCACCATGCTGGCTATCCGGGTGTTAGCTGAC CAAAATGTCATTCCTAATGTGGCTAATGTCACTTGCTATTACCAGCCAGCCCCCTATGTAGCAGATGCC AACTTTAGCAATTACTACATTGCACAGGTTCAGCCAGTATTCACGTGCCAGCAACAGACCTACTCCACT TGGCTACCCTGCAATTAA (SEQ ID NO: 15) G1P3 Entrez ID: 2537; OMIM: 147572; Uniprot ID: IFI6_HUMAN; ENSEMBL ID:  ENSG00000126709 ATGCGGCAGAAGGCGGTATCGCTTTTCTTGTGCTACCTGCTGCTCTTCACTTGCAGTGGGGTGGAGGCA GGTAAGAAAAAGTGCTCGGAGAGCTCGGACAGCGGCTCCGGGTTCTGGAAGGCCCTGACCTTCATGGCC GTCGGAGGAGGACTCGCAGTCGCCGGGCTGCCCGCGCTGGGCTTCACCGGCGCCGGCATCGCGGCCAAC TCGGTGGCTGCCTCGCTGATGAGCTGGTCTGCGATCCTGAATGGGGGCGGCGTGCCCGCCGGGGGGCTA GTGGCCACGCTGCAGAGCCTCGGGGCTGGTGGCAGCAGCGTCGTCATAGGTAATATTGGTGCCCTGATG GGCTACGCCACCCACAAGTATCTCGATAGTGAGGAGGATGAGGAGTAG (SEQ ID NO: 16) GNA12 Entrez ID: 2768; OMIM: 604394; Uniprot ID: GNA12_HUMAN; ENSEMBL ID: ENSG00000146535 ATGTCCGGGGTGGTGCGGACCCTCAGCCGCTGCCTGCTGCCGGCCGAGGCCGGCGGGGCCCGCGAGCGC AGGGCGGGCAGCGGCGCGCGCGACGCGGAGCGCGAGGCCCGGAGGCGTAGCCGCGACATCGACGCGCTG CTGGCCCGCGAGCGGCGCGCGGTCCGGCGCCTGGTGAAGATCCTGCTGCTGGGCGCGGGCGAGAGCGGC AAGTCCACGTTCCTCAAGCAGATGCGCATCATCCACGGCCGCGAGTTCGACCAGAAGGCGCTGCTGGAG TTCCGCGACACCATCTTCGACAACATCCTCAAGGGCTCAAGGGTTCTTGTTGATGCACGAGATAAGCTT GGCATTCCTTGGCAGTATTCTGAAAATGAGAAGCATGGGATGTTCCTGATGGCCTTCGAGAACAAGGCG GGGCTGCCTGTGGAGCCGGCCACCTTCCAGCTGTACGTCCCGGCCCTGAGCGCACTCTGGAGGGATTCT GGCATCAGGGAGGCTTTCAGCCGGAGAAGCGAGTTTCAGCTGGGGGAGTCGGTGAAGTACTTCCTGGAC AACTTGGACCGGATCGGCCAGCTGAATTACTTTCCTAGTAAGCAAGATATCCTGCTGGCTAGGAAAGCC ACCAAGGGAATTGTGGAGCATGACTTCGTTATTAAGAAGATCCCCTTTAAGATGGTGGATGTGGGCGGC CAGCGGTCCCAGCGCCAGAAGTGGTTCCAGTGCTTCGACGGGATCACGTCCATCCTGTTCATGGTCTCC TCCAGCGAGTACGACCAGGTCCTCATGGAGGACAGGCGCACCAACCGGCTGGTGGAGTCCATGAACATC TTCGAGACCATCGTCAACAACAAGCTCTTCTTCAACGTCTCCATCATTCTCTTCCTCAACAAGATGGAC CTCCTGGTGGAGAAGGTGAAGACCGTGAGCATCAAGAAGCACTTCCCGGACTTCAGGGGCGACCCGCAC AGGCTGGAGGACGTCCAGCGCTACCTGGTCCAGTGCTTCGACAGGAAGAGACGGAACCGCAGCAAGCCA CTCTTCCACCACTTCACCACCGCCATCGACACCGAGAACGTCCGCTTCGTGTTCCATGCTGTGAAAGAC ACCATCCTGCAGGAGAACCTGAAGGACATCATGCTGCAGTGA (SEQ ID NO: 17) HSPB1 Entrez ID: 3315; OMIM: 602195; Uniprot ID: HSPB1_RUMAN; ENSEMBL ID: ENSG00000106211 ATGACCGAGCGCCGCGTCCCCTTCTCGCTCCTGCGGGGCCCCAGCTGGGACCCCTTCCGCGACTGGTAC CCGCATAGCCGCCTCTTCGACCAGGCCTTCGGGCTGCCCCGGCTGCCGGAGGAGTGGTCGCAGTGGTTA GGCGGCAGCAGCTGGCCAGGCTACGTGCGCCCCCTGCCCCCCGCCGCCATCGAGAGCCCCGCAGTGGCC GCGCCCGCCTACAGCCGCGCGCTCAGCCGGCAACTCAGCAGCGGGGTCTCGGAGATCCGGCACACTGCG GACCGCTGGCGCGTGTCCCTGGATGTCAACCACTTCGCCCCGGACGAGCTGACGGTCAAGACCAAGGAT GGCGTGGTGGAGATCACCGGCAAGCACGAGGAGCGGCAGGACGAGCATGGCTACATCTCCCGGTGCTTC ACGCGGAAATACACGCTGCCCCCCGGTGTGGACCCCACCCAAGTTTCCTCCTCCCTGTCCCCTGAGGGC ACACTGACCGTGGAGGCCCCCATGCCCAAGCTAGCCACGCAGTCCAACGAGATCACCATCCCAGTCACC TTCGAGTCGCGGGCCCAGCTTGGGGGCCCAGAAGCTGCAAAATCCGATGAGACTGCCGCCAAGTAA (SEQ ID NO: 18) ID3 Entrez ID: 3399; OMIM: 600277; Uniprot ID: ID3_RUMAN; ENSEMBL ID:  ENSG00000117318 ATGAAGGCGCTGAGCCCGGTGCGCGGCTGCTACGAGGCGGTGTGCTGCCTGTCGGAACGCAGTCTGGCC ATCGCCCGGGGCCGAGGGAAGGGCCCGGCAGCTGAGGAGCCGCTGAGCTTGCTGGACGACATGAACCAC TGCTACTCCCGCCTGCGGGAACTGGTACCCGGAGTCCCGAGAGGCACTCAGCTTAGCCAGGTGGAAATC CTACAGCGCGTCATCGACTACATTCTCGACCTGCAGGTAGTCCTGGCCGAGCCAGCCCCTGGACCCCCT GATGGCCCCCACCTTCCCATCCAGACAGCCGAGCTCACTCCGGAACTTGTCATCTCCAACGACAAAAGG AGCTTTTGCCACTGA (SEQ ID NO: 19) IFITM2 Entrez ID: 10581; OMIM: 605578; Uniprot ID: IFM2_RUMAN; ENSEMBL ID: ENSG00000185201 ATGAACCACATTGTGCAAACCTTCTCTCCTGTCAACAGCGGCCAGCCTCCCAACTACGAGATGCTCAAG GAGGAGCAGGAAGTGGCTATGCTGGGGGTGCCCCACAACCCTGCTCCCCCGATGTCCACCGTGATCCAC ATCCGCAGCGAGACCTCCGTGCCTGACCATGTGGTCTGGTCCCTGTTCAACACCCTCTTCATGAACACC TGCTGCCTGGGCTTCATAGCATTCGCGTACTCCGTGAAGTCTAGGGACAGGAAGATGGTTGGCGACGTG ACCGGGGCCCAGGCCTATGCCTCCACCGCCAAGTGCCTGAACATCTGGGCCCTGATTTTGGGCATCTTC ATGACCATTCTGCTCATCATCATCCCAGTGTTGGTCGTCCAGGCCCAGCGATAG (SEQ ID NO: 20) ITPR1 Entrez ID: 3708; OMIM: 147265; Uniprot ID: ITPR1_RUMAN; ENSEMBL ID: ENSG00000150995 ATGTCTGACAAAATGTCTAGCTTCCTACATATTGGAGACATTTGTTCTCTGTACGCGGAGGGATCGACA AATGGATTTATTAGCACCTTGGGCCTGGTTGATGATCGTTGTGTTGTACAGCCAGAAACCGGGGACCTT AACAATCCACCTAAGAAATTCAGAGACTGCCTCTTTAAGCTATGTCCCATGAACCGCTACTCTGCCCAA AAGCAGTTCTGGAAAGCCGCTAAGCCTGGGGCCAACAGCACCACAGACGCAGTGCTACTCAACAAACTG CACCACGCTGCAGACTTGGAAAAGAAGCAGAATGAGACAGAAAACAGGAAATTGCTGGGGACCGTAATC CAGTATGGCAATGTGATCCAGCTCCTGCATTTGAAAAGTAATAAATACCTAACAGTGAATAAGAGGCTT CCTGCTCTGTTGGAGAAGAATGCCATGAGAGTCACATTGGACGAGGCTGGAAATGAAGGGTCCTGGTTT TATATTCAGCCATTCTACAAGCTGCGATCCATTGGAGACAGCGTGGTCATAGGTGACAAGGTGGTTCTG AACCCCGTCAATGCTGGTCAGCCCCTACATGCTAGCAGCCATCAACTGGTAGATAACCCAGGCTGCAAT GAGGTCAATTCCGTCAACTGCAATACAAGCTGGAAAATAGTCCTTTTCATGAAATGGAGTGATAACAAA GACGACATATTAAAGGGGGGTGACGTGGTGAGGCTGTTTCATGCTGAGCAGGAGAAGTTTCTCACCTGT GACGAACACAGGAAGAAGCAGCACGTCTTCCTGAGAACCACGGGCCGGCAGTCGGCCACATCTGCCACC AGTTCAAAAGCCCTGTGGGAGGTGGAGGTGGTCCAGCATGACCCATGTCGGGGCGGAGCAGGGTATTGG AACAGCCTTTTCCGTTTCAAGCATCTGGCCACGGGGCATTACTTGGCAGCAGAGGTAGACCCTGACTTT GAGGAAGAATGCCTGGAGTTTCAGCCCTCAGTGGACCCTGATCAGGACGCCTCTCGAAGTAGGTTGCGG AATGCCCAAGAAAAGATGGTATACTCCCTGGTCTCTGTGCCTGAAGGCAATGACATCTCCTCCATTTTC GAGCTAGATCCCACCACTCTGCGTGGAGGTGACAGCCTTGTCCCAAGGAACTCTTATGTTCGGCTCAGA CACCTATGTACTAATACCTGGGTTCACAGCACAAATATTCCTATTGACAAGGAAGAAGAAAAGCCCGTG ATGCTGAAAATTGGCACCTCTCCTGTGAAGGAGGATAAGGAAGCATTTGCCATAGTTCCGGTTTCTCCT GCTGAAGTTCGGGACCTGGACTTTGCCAATGATGCCAGCAAGGTGCTGGGCTCCATTGCTGGGAAGCTA GAGAAGGGCACCATCACCCAGAATGAAAGGAGGTCTGTAACCAAGCTGCTAGAAGATTTGGTTTACTTC GTCACTGGTGGAACTAATTCTGGTCAAGATGTTCTCGAAGTTGTCTTCTCCAAGCCCAACAGAGAACGG CAGAAACTGATGAGAGAACAGAATATTCTCAAGCAGATCTTCAAGTTGTTACAAGCCCCATTCACAGAC TGCGGTGATGGCCCAATGCTTCGGCTGGAAGAGCTCGGGGACCAGCGGCACGCTCCTTTCAGACACATC TGCCGGCTCTGCTACAGGGTGCTGAGACACTCGCAGCAAGACTACAGGAAGAACCAGGAGTATATAGCC AAGCAGTTTGGCTTCATGCAGAAGCAGATTGGCTATGATGTGTTGGCTGAAGACACTATCACTGCCCTG CTCCACAATAATCGGAAACTCCTGGAAAAACACATTACCGCGGCAGAGATTGACACATTTGTCAGCCTG GTGCGAAAGAACAGGGAGCCCAGATTCTTAGATTACCTCTCCGACCTCTGTGTCTCCATGAACAAATCA ATTCCAGTGACCCAGGAACTGATATGTAAAGCTGTGCTGAACCCCACCAACGCTGACATCCTGATTGAG ACCAAGTTGGTTCTTTCTCGTTTTGAATTTGAAGGTGTCTCTTCCACTGGAGAGAATGCTCTGGAGGCA GGAGAAGACGAGGAAGAGGTGTGGCTGTTTTGGAGGGACAGCAACAAAGAGATTCGCAGCAAGAGTGTG AGGGAATTGGCTCAGGATGCTAAAGAAGGGCAGAAGGAGGACCGAGACGTTCTCAGCTACTACAGATAT CAGCTGAACCTCTTTGCGAGGATGTGTCTGGACCGCCAATACCTGGCCATCAACGAAATCTCAGGCCAG CTGGATGTCGATCTCATTCTCCGCTGCATGTCTGACGAGAACCTGCCCTATGACCTCAGGGCGTCCTTC TGCCGCCTCATGCTTCACATGCATGTGGACCGAGATCCCCAGGAACAAGTCACCCCCGTGAAATATGCC CGCCTCTGGTCGGAGATTCCCTCGGAGATCGCCATTGACGACTATGATAGTAGTGGAGCTTCCAAAGAT GAAATTAAGGAGAGATTTGCTCAGACCATGGAGTTTGTGGAGGAGTATTTAAGAGATGTGGTTTGTCAG AGGTTCCCTTTCTCTGATAAAGAGAAGAATAAGCTTACGTTTGAGGTTGTAAATTTAGCTAGGAATCTC ATATACTTTGGTTTCTACAACTTCTCTGACCTTCTACGATTAACTAAGATCCTTCTGGCCATATTGGAC TGTGTACATGTGACAACAATCTTCCCCATTAGCAAGATGGCGAAAGGAGAAGAGAATAAAGGCAGTAAC GTGATGAGATCTATTCATGGCGTGGGAGAGCTGATGACCCAGGTGGTGCTCCGGGGAGGAGGCTTTTTG CCCATGACTCCCATGGCTGCTGCCCCTGAAGGCAATGTGAAGCAGGCAGAGCCTGAGAAGGAGGACATC ATGGTCATGGACACCAAGCTGAAGATCATTGAGATACTCCAGTTTATTTTGAATGTGAGGTTGGATTAT AGGATCTCCTGCCTCCTGTGTATATTTAAGCGAGAGTTTGATGAAAGCAATTCCCAGACTTCAGAAACA TCCTCCGGAAACAGCAGCCAAGAAGGGCCAAGTAATGTACCAGGTGCTCTTGACTTTGAACACATTGAA GAACAAGCAGAAGGCATCTTTGGAGGAAGTGAGGAGAACACCCCACTGGACTTGGATGACCACGGCGGC AGAACCTTTCTCCGTGTCCTGCTCCACTTGACGATGCATGACTACCCACCCCTGGTGTCAGGGGCCCTG CAGCTCCTCTTCCGGCACTTCAGCCAGAGGCAGGAGGTGCTCCAGGCCTTCAAACAGGTTCAACTGCTG GTTACCAGCCAAGATGTGGACAACTACAAACAGATCAAACAAGACTTGGATCAACTGAGGTCCATCGTG GAAAAGTCAGAGCTTTGGGTGTACAAAGGGCAGGGCCCCGATGAGACTATGGATGGTGCATCTGGAGAA AATGAACATAAGAAAACGGAGGAGGGAAATAACAAGCCACAAAAGCATGAAAGCACCAGCAGCTACAAC TACAGAGTGGTCAAAGAGATTTTGATTCGGCTTAGCAAACTCTGTGTTCAAGAGAGTGCCTCAGTGAGA AAGAGCAGGAAGCAGCAACAGCGTCTGCTCCGGAACATGGGCGCGCACGCCGTGGTGCTGGAGCTGCTG CAGATTCCCTATGAGAAGGCCGAAGATACCAAGATGCAAGAGATAATGAGGTTGGCTCATGAATTTTTG CAGAATTTCTGCGCAGGCAACCAGCAGAATCAAGCTTTGCTACATAAACACATAAACCTGTTTCTCAAC CCAGGGATCCTGGAGGCAGTAACCATGCAGCACATCTTCATGAACAATTTCCAGCTTTGCAGTGAGATC AACGAGAGAGTTGTTCAGCACTTCGTTCACTGCATAGAGACTCACGGTCGGAATGTCCAGTATATAAAG TTCTTACAGACAATTGTCAAGGCAGAAGGGAAATTTATTAAAAAATGCCAAGACATGGTTATGGCCGAG CTGGTCAATTCGGGAGAGGATGTCCTCGTGTTCTACAACGACAGAGCCTCTTTCCAGACTCTGATCCAG ATGATGCGGTCAGAACGGGATCGGATGGATGAGAACAGCCCTCTCATGTACCACATCCACTTGGTCGAG CTCCTGGCTGTGTGCACGGAGGGTAAGAATGTCTACACAGAGATCAAGTGCAACTCCCTGCTCCCGCTG GATGACATCGTTCGCGTGGTGACCCACGAGGACTGCATCCCTGAGGTTAAAATTGCATACATTAACTTC CTGAATCACTGCTATGTGGATACAGAGGTGGAAATGAAGGAGATTTATACCAGCAATCACATGTGGAAA TTGTTTGAGAATTTCCTTGTAGACATCTGCAGGGCCTGTAACAACACTAGTGACAGGAAACATGCAGAC TCGATTTTGGAGAAGTATGTCACCGAAATCGTCATGAGTATTGTTACTACTTTCTTCAGCTCTCCCTTC TCAGACCAGAGTACGACTTTGCAGACTCGCCAGCCTGTCTTTGTGCAACTGCTGCAAGGCGTGTTCAGG GTTTACCACTGCAACTGGTTAATGCCAAGCCAAAAAGCCTCCGTGGAGAGCTGTATTCGGGTGCTGTCT GATGTAGCCAAGAGCCGGGCCATTGCCATTCCCGTGGACCTGGACAGCCAAGTCAACAACCTCTTTCTC AAGTCCCACAGCATTGTGCAGAAAACAGCCATGAACTGGCGGCTCTCAGCCCGCAATGCCGCACGCAGG GACTCTGTTCTGGCAGCTTCCAGAGACTACCGGAATATCATTGAGAGATTGCAGGACATCGTCTCCGCG CTGGAGGACCGTCTCAGGCCCCTGGTGCAGGCAGAGTTATCTGTGCTCGTGGATGTTCTCCACAGACCC GAGCTGCTTTTCCCAGAGAACACAGACGCCAGAAGGAAATGTGAAAGTGGCGGTTTCATTTGCAAGTTA ATAAAGCATACAAAACAGCTGCTAGAAGAAAATGAAGAGAAGCTCTGCATTAAGGTCCTACAGACCCTG AGGGAAATGATGACCAAAGATAGAGGCTATGGAGAAAAGGGTGAGGCGCTCAGGCAAGTTCTGGTCAAC CGTTACTATGGAAACGTCAGACCTTCGGGACGAAGAGAGAGCCTTACCAGCTTTGGCAATGGCCCACTG TCAGCAGGAGGACCCGGCAAGCCCGGGGGAGGAGGGGGAGGTTCCGGATCCAGCTCTATGAGCAGGGGT GAGATGAGTCTGGCCGAGGTTCAGTGTCACCTTGACAAGGAGGGGGCTTCCAATCTAGTTATCGACCTC ATCATGAACGCATCCAGTGACCGAGTGTTCCATGAAAGCATTCTCCTGGCCATTGCCCTTCTGGAAGGA GGCAACACCACCATCCAGCACTCCTTTTTCTGTCGCTTGACAGAAGATAAGAAGTCAGAGAAATTCTTT AAGGTGTTTTATGACCGGATGAAGGTGGCCCAGCAAGAAATCAAAGCAACAGTGACAGTGAACACCAGT GACTTGGGAAATAAAAAGAAAGACGATGAGGTAGACAGGGATGCCCCATCACGGAAAAAAGCTAAAGAG CCCACAACACAGATAACAGAAGAGGTCCGGGATCAGCTCCTGGAGGCCTCCGCTGCCACCAGGAAAGCC TTCACCACTTTCAGGAGGGAGGCTGATCCCGACGACCACTACCAGCCTGGAGAGGGCACCCAGGCCACT GCCGACAAGGCCAAGGACGACCTGGAGATGAGCGCGGTCATCACCATCATGCAGCCCATCCTCCGCTTC CTTCAGCTCCTGTGTGAAAACCACAACCGAGACCTGCAGAACTTCCTCCGTTGCCAAAATAACAAGACC AACTACAATTTGGTATGTGAGACCCTGCAGTTTCTGGACTGTATTTGTGGAAGCACAACTGGAGGCCTT GGTCTTCTGGGCTTGTATATAAATGAAAAGAACGTAGCGCTTATCAACCAAACCCTGGAAAGTCTGACC GAATACTGTCAAGGACCTTGCCATGAGAACCAGAACTGCATAGCCACCCATGAATCCAATGGCATTGAC ATCATCACAGCCCTGATCCTCAATGATATCAATCCTTTGGGAAAGAAGAGGATGGACCTTGTGTTAGAA CTGAAGAACAATGCCTCGAAGTTGCTCCTGGCCATCATGGAAAGCAGGCACGACAGTGAAAACGCAGAG AGGATACTTTATAACATGAGGCCCAAGGAACTGGTGGAAGTGATCAAGAAAGCCTACATGCAAGGTGAA GTGGAATTTGAGGATGGAGAAAACGGTGAGGATGGGGCGGCGTCCCCCAGGAACGTGGGGCACAACATC TACATATTAGCCCATCAGTTGGCTCGGCATAACAAAGAACTTCAGAGCATGCTGAAACCTGGTGGCCAA GTGGACGGAGATGAAGCCCTGGAGTTTTATGCCAAGCACACGGCGCAGATAGAGATTGTCAGATTAGAC CGAACAATGGAACAGATAGTCTTTCCCGTGCCCAGCATATGTGAATTCCTAACCAAGGAGTCAAAACTA CGAATTTACTATACTACAGAGAGAGACGAACAAGGCAGCAAAATCAATGATTTCTTTCTGCGGTCTGAA GACCTCTTCAATGAAATGAATTGGCAGAAGAAACTGAGAGCCCAGCCCGTGTTGTACTGGTGTGCCCGC AACATGTCTTTCTGGAGCAGCATTTCGTTTAACCTGGCCGTCCTGATGAACCTGCTGGTGGCGTTTTTC TACCCGTTTAAGGGAGTCCGAGGAGGAACCCTGGAGCCCCACTGGTCGGGACTCCTGTGGACAGCCATG CTCATCTCTCTGGCCATCGTCATTGCCCTCCCCAAGCCCCATGGCATCCGGGCCTTAATTGCCTCCACA ATTCTACGACTGATATTTTCAGTCGGGTTACAACCCACGTTGTTTCTTCTGGGCGCTTTCAATGTATGC AATAAAATCATCTTTCTAATGAGCTTTGTGGGCAACTGTGGGACATTCACAAGAGGCTACCGAGCCATG GTTCTGGATGTTGAGTTCCTCTATCATTTGTTGTATCTGGTGATCTGTGCCATGGGGCTCTTTGTCCAT GAATTCTTCTACAGTCTGCTGCTTTTTGATTTAGTGTACAGAGAAGAGACTTTGCTTAATGTCATTAAA AGTGTCACTCGCAATGGACGGTCCATCATCCTGACAGCAGTTCTGGCTCTGATCCTCGTTTACCTGTTC TCAATAGTGGGCTATCTTTTCTTCAAGGATGACTTTATCTTGGAAGTAGATAGGCTGCCCAATGAAACA GCTGTTCCAGAAACCGGCGAGAGTTTGGCAAGCGAGTTCCTGTTCTCCGATGTGTGTAGGGTGGAGAGT GGGGAGAACTGCTCCTCTCCTGCACCCAGAGAAGAGCTGGTCCCTGCAGAAGAGACGGAACAGGATAAA GAGCACACATGTGAGACGCTGCTGATGTGCATTGTCACTGTGCTGAGTCACGGGCTGCGGAGCGGGGGT GGAGTAGGAGATGTACTCAGGAAGCCGTCCAAAGAGGAACCCCTGTTTGCTGCTAGAGTTATTTATGAC CTCTTGTTCTTCTTCATGGTCATCATCATTGTTCTTAACCTGATTTTTGGGGTTATCATTGACACTTTT GCTGACCTGAGGAGTGAGAAGCAGAAGAAGGAAGAGATCTTGAAGACCACGTGCTTTATCTGTGGCTTG GAAAGAGACAAGTTTGACAACAAGACTGTCACCTTTGAAGAGCACATCAAGGAAGAACACAACATGTGG CACTATCTGTGCTTCATCGTCCTGGTGAAAGTAAAGGACTCCACCGAATATACTGGGCCTGAGAGTTAC GTGGCAGAAATGATCAAGGAAAGAAACCTTGACTGGTTCCCCAGGATGAGAGCCATGTCATTGGTCAGC AGTGATTCTGAAGGAGAACAGAATGAGCTGAGAAACCTGCAGGAGAAGCTGGAGTCCACCATGAAACTT GTCACGAACCTTTCTGGCCAGCTGTCGGAATTAAAGGATCAGATGACAGAACAAAGGAAGCAGAAACAA AGAATTGGTCTTCTAGGACATCCTCCTCACATGAATGTCAACCCACAACAACCAGCATAA (SEQ ID NO: 21) JUN Entrez ID: 3725; OMIM: 165160; Uniprot ID: JUN_HUMAN; ENSEMBL ID:  ENSG00000177606 ATGACTGCAAAGATGGAAACGACCTTCTATGACGATGCCCTCAACGCCTCGTTCCTCCCGTCCGAGAGC GGACCTTATGGCTACAGTAACCCCAAGATCCTGAAACAGAGCATGACCCTGAACCTGGCCGACCCAGTG GGGAGCCTGAAGCCGCACCTCCGCGCCAAGAACTCGGACCTCCTCACCTCGCCCGACGTGGGGCTGCTC AAGCTGGCGTCGCCCGAGCTGGAGCGCCTGATAATCCAGTCCAGCAACGGGCACATCACCACCACGCCG ACCCCCACCCAGTTCCTGTGCCCCAAGAACGTGACAGATGAGCAGGAGGGCTTCGCCGAGGGCTTCGTG CGCGCCCTGGCCGAACTGCACAGCCAGAACACGCTGCCCAGCGTCACGTCGGCGGCGCAGCCGGTCAAC GGGGCAGGCATGGTGGCTCCCGCGGTAGCCTCGGTGGCAGGGGGCAGCGGCAGCGGCGGCTTCAGCGCC AGCCTGCACAGCGAGCCGCCGGTCTACGCAAACCTCAGCAACTTCAACCCAGGCGCGCTGAGCAGCGGC GGCGGGGCGCCCTCCTACGGCGCGGCCGGCCTGGCCTTTCCCGCGCAACCCCAGCAGCAGCAGCAGCCG CCGCACCACCTGCCCCAGCAGATGCCCGTGCAGCACCCGCGGCTGCAGGCCCTGAAGGAGGAGCCTCAG ACAGTGCCCGAGATGCCCGGCGAGACACCGCCCCTGTCCCCCATCGACATGGAGTCCCAGGAGCGGATC AAGGCGGAGAGGAAGCGCATGAGGAACCGCATCGCTGCCTCCAAGTGCCGAAAAAGGAAGCTGGAGAGA ATCGCCCGGCTGGAGGAAAAAGTGAAAACCTTGAAAGCTCAGAACTCGGAGCTGGCGTCCACGGCCAAC ATGCTCAGGGAACAGGTGGCACAGCTTAAACAGAAAGTCATGAACCACGTTAACAGTGGGTGCCAACTC ATGCTAACGCAGCAGTTGCAAACATTTTGA (SEQ ID NO: 22) LY96 Entrez ID: 23643; OMIM: 605243; Uniprot ID: LY96_HUMAN; ENSEMBL ID: ENSG00000154589 ATGTTACCATTTCTGTTTTTTTCCACCCTGTTTTCTTCCATATTTACTGAAGCTCAGAAGCAGTATTGG GTCTGCAACTCATCCGATGCAAGTATTTCATACACCTACTGTGGGAGAGATTTAAAGCAATTATATTTC AATCTCTATATAACTGTCAACACCATGAATCTTCCAAAGCGCAAAGAAGTTATTTGCCGAGGATCTGAT GACGATTACTCTTTTTGCAGAGCTCTGAAGGGAGAGACTGTGAATACAACAATATCATTCTCCTTCAAG GGAATAAAATTTTCTAAGGGAAAATACAAATGTGTTGTTGAAGCTATTTCTGGGAGCCCAGAAGAAATG CTCTTTTGCTTGGAGTTTGTCATCCTACACCAACCTAATTCAAATTAG (SEQ ID NO: 23) MAP2K1 Entrez ID: 5604; OMIM: 176872; Uniprot ID: MP2K1_HUMAN; ENSEMBL ID: ENSG00000169032 ATGCCCAAGAAGAAGCCGACGCCCATCCAGCTGAACCCGGCCCCCGACGGCTCTGCAGTTAACGGGACC AGCTCTGCGGAGACCAACTTGGAGGCCTTGCAGAAGAAGCTGGAGGAGCTAGAGCTTGATGAGCAGCAG CGAAAGCGCCTTGAGGCCTTTCTTACCCAGAAGCAGAAGGTGGGAGAACTGAAGGATGACGACTTTGAG AAGATCAGTGAGCTGGGGGCTGGCAATGGCGGTGTGGTGTTCAAGGTCTCCCACAAGCCTTCTGGCCTG GTCATGGCCAGAAAGCTAATTCATCTGGAGATCAAACCCGCAATCCGGAACCAGATCATAAGGGAGCTG CAGGTTCTGCATGAGTGCAACTCTCCGTACATCGTGGGCTTCTATGGTGCGTTCTACAGCGATGGCGAG ATCAGTATCTGCATGGAGCACATGGATGGAGGTTCTCTGGATCAAGTCCTGAAGAAAGCTGGAAGAATT CCTGAACAAATTTTAGGAAAAGTTAGCATTGCTGTAATAAAAGGCCTGACATATCTGAGGGAGAAGCAC AAGATCATGCACAGAGATGTCAAGCCCTCCAACATCCTAGTCAACTCCCGTGGGGAGATCAAGCTCTGT GACTTTGGGGTCAGCGGGCAGCTCATCGACTCCATGGCCAACTCCTTCGTGGGCACAAGGTCCTACATG TCGCCAGAAAGACTCCAGGGGACTCATTACTCTGTGCAGTCAGACATCTGGAGCATGGGACTGTCTCTG GTAGAGATGGCGGTTGGGAGGTATCCCATCCCTCCTCCAGATGCCAAGGAGCTGGAGCTGATGTTTGGG TGCCAGGTGGAAGGAGATGCGGCTGAGACCCCACCCAGGCCAAGGACCCCCGGGAGGCCCCTTAGCTCA TACGGAATGGACAGCCGACCTCCCATGGCAATTTTTGAGTTGTTGGATTACATAGTCAACGAGCCTCCT CCAAAACTGCCCAGTGGAGTGTTCAGTCTGGAATTTCAAGATTTTGTGAATAAATGCTTAATAAAAAAC CCCGCAGAGAGAGCAGATTTGAAGCAACTCATGGTTCATGCTTTTATCAAGAGATCTGATGCTGAGGAA GTGGATTTTGCAGGTTGGCTCTGCTCCACCATCGGCCTTAACCAGCCCAGCACACCAACCCATGCTGCT GGCGTCTAA (SEQ ID NO: 24) MCL1 Entrez ID: 7249; OMIM: 191092; Uniprot ID: TSC2_HUMAN; ENSEMBL ID:  ENSG00000103197 ATGTTTGGCCTCAAAAGAAACGCGGTAATCGGACTCAACCTCTACTGTGGGGGGGCCGGCTTGGGGGCC GGCAGCGGCGGCGCCACCCGCCCGGGAGGGCGACTTTTGGCCACCGGCGCCAAGGACACAAAGCCAATG GGCAGGTCTGGGGCCACCAGCAGGAAGGCGCTGGAGACCTTACGACGGGTTGGGGATGGCGTGCAGCGC AACCACGAGACGGCCTTCCAAGGCATGCTTCGGAAACTGGACATCAAAAACGAAGACGATGTGAAATCG TTGTCTCGAGTGATGATCCATGTTTTCAGCGACGGCGTAACAAACTGGGGCAGGATTGTGACTCTCATT TCTTTTGGTGCCTTTGTGGCTAAACACTTGAAGACCATAAACCAAGAAAGCTGCATCGAACCATTAGCA GAAAGTATCACAGACGTTCTCGTAAGGACAAAACGGGACTGGCTAGTTAAACAAAGAGGCTGGGATGGG TTTGTGGAGTTCTTCCATGTAGAGGACCTAGAAGGTGGCATCAGGAATGTGCTGCTGGCTTTTGCAGGT GTTGCTGGAGTAGGAGCTGGTTTGGCATATCTAATAAGATAG (SEQ ID NO: 25) MT2A Entrez ID: 4502; OMIM: 156360; Uniprot ID: MT2_HUMAN; ENSEMBL ID:  ENSG00000125148 ATGGATCCCAACTGCTCCTGCGCCGCCGGTGACTCCTGCACCTGCGCCGGCTCCTGCAAATGCAAAGAG TGCAAATGCACCTCCTGCAAGAAAAGCTGCTGCTCCTGCTGCCCTGTGGGCTGTGCCAAGTGTGCCCAG GGCTGCATCTGCAAAGGGGCGTCGGACAAGTGCAGCTGCTGCGCCTGA (SEQ ID NO: 26) NEFM Entrez ID: 4741; OMIM: 162250; Uniprot ID: NFM_HUMAN; ENSEMBL ID:  ENSG00000104722 ATGGCTCGTCATTTGCGCGAATACCAGGACCTCCTCAACGTCAAGATGGCTCTGGATATAGAAATCGCT GCGTACAGAAAACTCCTGGAGGGTGAAGAGACTAGATTTAGCACATTTGCAGGAAGCATCACTGGGCCA CTGTATACACACCGACCCCCAATCACAATATCCAGTAAGATTCAGAAACCCAAGGTGGAAGCTCCCAAG CTTAAGGTCCAACACAAATTTGTCGAGGAGATCATAGAGGAAACCAAAGTGGAGGATGAGAAGTCAGAA ATGGAAGAGGCCCTGACAGCCATTACAGAGGAATTGGCCGTTTCCATGAAGGAAGAGAAGAAAGAAGCA GCAGAAGAAAAGGAAGAGGAACCCGAAGCTGAAGAAGAAGAAGTAGCTGCCAAAAAGTCTCCAGTGAAA GCAACTGCACCTGAAGTTAAAGAAGAGGAAGGGGAAAAGGAGGAAGAAGAAGGCCAGGAAGAAGAGGAG GAAGAAGATGAGGGAGCTAAGTCAGACCAAGCCGAAGAGGGAGGATCCGAGAAGGAAGGCTCTAGTGAA AAAGAGGAAGGTGAGCAGGAAGAAGGAGAAACAGAAGCTGAAGCTGAAGGAGAGGAAGCCGAAGCTAAA GAGGAAAAGAAAGTGGAGGAAAAGAGTGAGGAAGTGGCTACCAAGGAGGAGCTGGTGGCAGATGCCAAG GTGGAAAAGCCAGAAAAAGCCAAGTCTCCTGTGCCAAAATCACCAGTGGAAGAGAAAGGCAAGTCTCCT GTGCCCAAGTCACCAGTGGAAGAGAAAGGCAAGTCTCCTGTGCCCAAGTCACCAGTGGAAGAGAAAGGC AAGTCTCCTGTGCCGAAATCACCAGTGGAAGAGAAAGGCAAGTCTCCTGTGTCAAAATCACCAGTGGAA GAGAAAGCCAAATCTCCTGTGCCAAAATCACCAGTGGAAGAGGCAAAGTCAAAAGCAGAAGTGGGGAAA GGTGAACAGAAAGAGGAAGAAGAAAAGGAAGTCAAGGAAGCTCCCAAGGAAGAGAAGGTAGAGAAAAAG GAAGAGAAACCAAAGGATGTGCCAGAGAAGAAGAAAGCTGAGTCCCCTGTAAAGGAGGAAGCTGTGGCA GAGGTGGTCACCATCACCAAATCGGTAAAGGTGCACTTGGAGAAAGAGACCAAAGAAGAGGGGAAGCCA CTGCAGCAGGAGAAAGAGAAGGAGAAAGCGGGAGGAGAGGGAGGAAGTGAGGAGGAAGGGAGTGATAAA GGTGCCAAGGGATCCAGGAAGGAAGACATAGCTGTCAATGGGGAGGTAGAAGGAAAAGAGGAGGTAGAG CAGGAGACCAAGGAAAAAGGCAGTGGGAGGGAAGAGGAGAAAGGCGTTGTCACCAATGGCCTAGACTTG AGCCCAGCAGATGAAAAGAAGGGGGGTGATAAAAGTGAGGAGAAAGTGGTGGTGACCAAAACGGTAGAA AAAATCACCAGTGAGGGGGGAGATGGTGCTACCAAATACATCACTAAATCTGTAACCGTCACTCAAAAG GTTGAAGAGCATGAAGAGACCTTTGAGGAGAAACTAGTGTCTACTAAAAAGGTAGAAAAAGTCACTTCA CACGCCATAGTAAAGGAAGTCACCCAGAGTGACTAA (SEQ ID NO: 27) NQO1 Entrez ID: 1728; OMIM: 125860; Uniprot ID: NQO1_HUMAN; ENSEMBL ID:  ENSG00000181019 ATGGTCGGCAGAAGAGCACTGATCGTACTGGCTCACTCAGAGAGGACGTCCTTCAACTATGCCATGAAG GAGGCTGCTGCAGCGGCTTTGAAGAAGAAAGGATGGGAGGTGGTGGAGTCGGACCTCTATGCCATGAAC TTCAATCCCATCATTTCCAGAAAGGACATCACAGGTAAACTGAAGGACCCTGCGAACTTTCAGTATCCT GCCGAGTCTGTTCTGGCTTATAAAGAAGGCCATCTGAGCCCAGATATTGTGGCTGAACAAAAGAAGCTG GAAGCCGCAGACCTTGTGATATTCCAGTTCCCCCTGCAGTGGTTTGGAGTCCCTGCCATTCTGAAAGGC TGGTTTGAGCGAGTGTTCATAGGAGAGTTTGCTTACACTTACGCTGCCATGTATGACAAAGGACCCTTC CGGAGTAAGAAGGCAGTGCTTTCCATCACCACTGGTGGCAGTGGCTCCATGTACTCTCTGCAAGGGATC CACGGGGACATGAATGTCATTCTCTGGCCAATTCAGAGTGGCATTCTGCATTTCTGTGGCTTCCAAGTC TTAGAACCTCAACTGACATATAGCATTGGGCACACTCCAGCAGACGCCCGAATTCAAATCCTGGAAGGA TGGAAGAAACGCCTGGAGAATATTTGGGATGAGACACCACTGTATTTTGCTCCAAGCAGCCTCTTTGAC CTAAACTTCCAGGCAGGATTCTTAATGAAAAAAGAGGTACAGGATGAGGAGAAAAACAAGAAATTTGGC CTTTCTGTGGGCCATCACTTGGGCAAGTCCATCCCAACTGACAACCAGATCAAAGCTAGAAAATGA (SEQ ID NO: 28) P4HA1 Entrez ID: 5033; OMIM: 176710; Uniprot ID: P4HA1_HUMAN; ENSEMBL ID: ENSG00000122884 ATGATCTGGTATATATTAATTATAGGAATTCTGCTTCCCCAGTCTTTGGCTCATCCAGGCTTTTTTACT TCAATTGGTCAGATGACTGATTTGATCCATACTGAGAAAGATCTGGTGACTTCTCTGAAAGATTATATT AAGGCAGAAGAGGACAAGTTAGAACAAATAAAAAAATGGGCAGAGAAGTTAGATCGGCTAACTAGTACA GCGACAAAAGATCCAGAAGGATTTGTTGGGCATCCAGTAAATGCATTCAAATTAATGAAACGTCTGAAT ACTGAGTGGAGTGAGTTGGAGAATCTGGTCCTTAAGGATATGTCAGATGGCTTTATCTCTAACCTAACC ATTCAGAGACAGTACTTTCCTAATGATGAAGATCAGGTTGGGGCAGCCAAAGCTCTGTTACGTCTCCAG GATACCTACAATTTGGATACAGATACCATCTCAAAGGGTAATCTTCCAGGAGTGAAACACAAATCTTTT CTAACGGCTGAGGACTGCTTTGAGTTGGGCAAAGTGGCCTATACAGAAGCAGATTATTACCATACGGAA CTGTGGATGGAACAAGCCCTAAGGCAACTGGATGAAGGCGAGATTTCTACCATAGATAAAGTCTCTGTT CTAGATTATTTGAGCTATGCGGTATATCAGCAGGGAGACCTGGATAAGGCACTTTTGCTCACAAAGAAG CTTCTTGAACTAGATCCTGAACATCAGAGAGCTAATGGTAACTTAAAATATTTTGAGTATATAATGGCT AAAGAAAAAGATGTCAATAAGTCTGCTTCAGATGACCAATCTGATCAGAAAACTACACCAAAGAAAAAA GGGGTTGCTGTGGATTACCTGCCAGAGAGACAGAAGTACGAAATGCTGTGCCGTGGGGAGGGTATCAAA ATGACCCCTCGGAGACAGAAAAAACTCTTTTGCCGCTACCATGATGGAAACCGTAATCCTAAATTTATT CTGGCTCCAGCTAAACAGGAGGATGAATGGGACAAGCCTCGTATTATTCGCTTCCATGATATTATTTCT GATGCAGAAATTGAAATCGTCAAAGACCTAGCAAAACCAAGGCTGAGCCGAGCTACAGTACATGACCCT GAGACTGGAAAATTGACCACAGCACAGTACAGAGTATCTAAGAGTGCCTGGCTCTCTGGCTATGAAAAT CCTGTGGTGTCTCGAATTAATATGAGAATACAAGATCTAACAGGACTAGATGTTTCCACAGCAGAGGAA TTACAGGTAGCAAATTATGGAGTTGGAGGACAGTATGAACCCCATTTTGACTTTGCACGGAAAGATGAG CCAGATGCTTTCAAAGAGCTGGGGACAGGAAATAGAATTGCTACATGGCTGTTTTATATGAGTGATGTG TCTGCAGGAGGAGCCACTGTTTTTCCTGAAGTTGGAGCTAGTGTTTGGCCCAAAAAAGGAACTGCTGTT TTCTGGTATAATCTGTTTGCCAGTGGAGAAGGAGATTATAGTACACGGCATGCAGCCTGTCCAGTGCTA GTTGGCAACAAATGGGTATCCAATAAATGGCTCCATGAACGTGGACAAGAATTTCGAAGACCTTGTACG TTGTCAGAATTGGAATGA (SEQ ID NO: 29) PLOD2 Entrez ID: 5352; OMIM: 601865; Uniprot ID: PLOD2_HUMAN; ENSEMBL ID: ENSG00000152952 ATGGGGGGATGCACGGTGAAGCCTCAGCTGCTGCTCCTGGCGCTCGTCCTCCACCCCTGGAATCCCTGT CTGGGTGCGGACTCGGAGAAGCCCTCGAGCATCCCCACAGATAAATTATTAGTCATAACTGTAGCAACA AAAGAAAGTGATGGATTCCATCGATTTATGCAGTCAGCCAAATATTTCAATTATACTGTGAAGGTCCTT GGTCAAGGAGAAGAATGGAGAGGTGGTGATGGAATTAATAGTATTGGAGGGGGCCAGAAAGTGAGATTA ATGAAAGAAGTCATGGAACACTATGCTGATCAAGATGATCTGGTTGTCATGTTTACTGAATGCTTTGAT GTCATATTTGCTGGTGGTCCAGAAGAAGTTCTAAAAAAATTCCAAAAGGCAAACCACAAAGTGGTCTTT GCAGCAGATGGAATTTTGTGGCCAGATAAAAGACTAGCAGACAAGTATCCTGTTGTGCACATTGGGAAA CGCTATCTGAATTCAGGAGGATTTATTGGCTATGCTCCATATGTCAACCGTATAGTTCAACAATGGAAT CTCCAGGATAATGATGATGATCAGCTCTTTTACACTAAAGTTTACATTGATCCACTGAAAAGGGAAGCT ATTAACATCACATTGGATCACAAATGCAAAATTTTCCAGACCTTAAATGGAGCTGTAGATGAAGTTGTT TTAAAATTTGAAAATGGCAAAGCCAGAGCTAAGAATACATTTTATGAAACATTACCAGTGGCAATTAAT GGAAATGGACCCACCAAGATTCTCCTGAATTATTTTGGAAACTATGTACCCAATTCATGGACACAGGAT AATGGCTGCACTCTTTGTGAATTCGATACAGTCGACTTGTCTGCAGTAGATGTCCATCCAAACGTATCA ATAGGTGTTTTTATTGAGCAACCAACCCCTTTTCTACCTCGGTTTCTGGACATATTGTTGACACTGGAT TACCCAAAAGAAGCACTTAAACTTTTTATTCATAACAAAGAAGTTTATCATGAAAAGGACATCAAGGTA TTTTTTGATAAAGCTAAGCATGAAATCAAAACTATAAAAATAGTAGGACCAGAAGAAAATCTAAGTCAA GCGGAAGCCAGAAACATGGGAATGGACTTTTGCCGTCAGGATGAAAAGTGTGATTATTACTTTAGTGTG GATGCAGATGTTGTTTTGACAAATCCAAGGACTTTAAAAATTTTGATTGAACAAAACAGAAAGATCATT GCTCCTCTTGTAACTCGTCATGGAAAGCTGTGGTCCAATTTCTGGGGAGCATTGAGTCCTGATGGATAC TATGCACGATCTGAAGATTATGTGGATATTGTTCAAGGGAATAGAGTAGGAGTATGGAATGTCCCATAT ATGGCTAATGTGTACTTAATTAAAGGAAAGACACTCCGATCAGAGATGAATGAAAGGAACTATTTTGTT CGTGATAAACTGGATCCTGATATGGCTCTTTGCCGAAATGCTAGAGAAATGGGTGTATTTATGTACATT TCTAATAGACATGAATTTGGAAGGCTATTATCCACTGCTAATTACAATACTTCCCATTATAACAATGAC CTCTGGCAGATTTTTGAAAATCCTGTGGACTGGAAGGAAAAGTATATAAACCGTGATTATTCAAAGATT TTCACTGAAAATATAGTTGAACAGCCCTGTCCAGATGTCTTTTGGTTCCCCATATTTTCTGAAAAAGCC TGTGATGAATTGGTAGAAGAAATGGAACATTACGGCAAATGGTCTGGGGGAAAACATCATGATAGCCGT ATATCTGGTGGTTATGAAAATGTCCCAACTGATGATATCCACATGAAGCAAGTTGATCTGGAGAATGTA TGGCTTCATTTTATCCGGGAGTTCATTGCACCAGTTACACTGAAGGTCTTTGCAGGCTATTATACGAAG GGATTTGCACTACTGAATTTTGTAGTAAAATACTCCCCTGAACGACAGCGTTCTCTTCGTCCTCATCAT GATGCTTCTACATTTACCATAAACATTGCACTTAATAACGTGGGAGAAGACTTTCAGGGAGGTGGTTGC AAATTTCTAAGGTACAATTGCTCTATTGAGTCACCACGAAAAGGCTGGAGCTTCATGCATCCTGGGAGA CTCACACATTTGCATGAAGGACTTCCTGTTAAAAATGGAACAAGATACATTGCAGTGTCATTTATAGAT CCCTAA (SEQ ID NO: 30) PLTP Entrez ID: 5360; OMIM: 172425; Uniprot ID: PLTP_HUMAN; ENSEMBL ID:  ENSG00000100979 ATGGCCCTCTTCGGGGCCCTCTTCCTAGCGCTGCTGGCAGGCGCACATGCAGAGTTCCCAGGCTGCAAG ATCCGCGTCACCTCCAAGGCGCTGGAGCTGGTGAAGCAGGAGGGGCTGCGCTTTCTGGAGCAAGAGCTG GAGACTATCACCATTCCGGACCTGCGGGGCAAAGAAGGCCACTTCTACTACAACATCTCTGAGGTGAAG GTCACAGAGCTGCAACTGACATCTTCCGAGCTCGATTTCCAGCCACAGCAGGAGCTGATGCTTCAAATC ACCAATGCCTCCTTGGGGCTGCGCTTCCGGAGACAGCTGCTCTACTGGTTCTTCTATGATGGGGGCTAC ATCAACGCCTCAGCTGAGGGTGTGTCCATCCGCACTGGTCTGGAGCTCTCCCGGGATCCCGCTGGACGG ATGAAAGTGTCCAATGTCTCCTGCCAGGCCTCTGTCTCCAGAATGCACGCGGCCTTCGGGGGAACCTTC AAGAAGGTGTATGATTTTCTCTCCACGTTCATCACCTCAGGGATGCGCTTCCTCCTCAACCAGCAGATC TGCCCTGTCCTCTACCACGCAGGGACGGTCCTGCTCAACTCCCTCCTGGACACCGTGCCTGTGCGCAGT TCTGTGGACGAGCTTGTTGGCATTGACTATTCCCTCATGAAGGATCCTGTGGCTTCCACCAGCAACCTG GACATGGACTTCCGGGGGGCCTTCTTCCCCCTGACTGAGAGGAACTGGAGCCTCCCCAACCGGGCAGTG GAGCCCCAGCTGCAGGAGGAAGAGCGGATGGTGTATGTGGCCTTCTCTGAGTTCTTCTTCGACTCTGCC ATGGAGAGCTACTTCCGGGCGGGGGCCCTGCAGCTGTTGCTGGTGGGGGACAAGGTGCCCCACGACCTG GACATGCTGCTGAGGGCCACCTACTTTGGGAGCATTGTCCTGCTGAGCCCAGCAGTGATTGACTCCCCA TTGAAGCTGGAGCTGCGGGTCCTGGCCCCACCGCGCTGCACCATCAAGCCCTCTGGCACCACCATCTCT GTCACTGCTAGCGTCACCATTGCCCTGGTCCCACCAGACCAGCCTGAGGTCCAGCTGTCCAGCATGACT ATGGACGCCCGTCTCAGCGCCAAGATGGCTCTCCGGGGGAAGGCCCTGCGCACGCAGCTGGACCTGCGC AGGTTCCGAATCTATTCCAACCATTCTGCACTGGAGTCGCTGGCTCTGATCCCATTACAGGCCCCTCTG AAGACCATGCTGCAGATTGGGGTGATGCCCATGCTCAATGAGCGGACCTGGCGTGGGGTGCAGATCCCA CTACCTGAGGGCATCAACTTTGTGCATGAGGTGGTGACGAACCATGCGGGATTCCTCACCATCGGGGCT GATCTCCACTTTGCCAAAGGGCTGCGAGAGGTGATTGAGAAGAACCGGCCTGCTGATGTCAGGGCGTCC ACTGCCCCCACACCGTCCACAGCAGCTGTCTGA (SEQ ID NO: 31) PTBP1 Entrez ID: 5725; OMIM: 600693; Uniprot ID: PTBP1_HUMAN; ENSEMBL ID: ENSG00000011304 ATGGACGGCATTGTCCCAGATATAGCCGTTGGTACAAAGCGGGGATCTGACGAGCTTTTCTCTACTTGT GTCACTAACGGACCGTTTATCATGAGCAGCAACTCGGCTTCTGCAGCAAACGGAAATGACAGCAAGAAG TTCAAAGGTGACAGCCGAAGTGCAGGCGTCCCCTCTAGAGTGATCCACATCCGGAAGCTCCCCATCGAC GTCACGGAGGGGGAAGTCATCTCCCTGGGGCTGCCCTTTGGGAAGGTCACCAACCTCCTGATGCTGAAG GGGAAAAACCAGGCCTTCATCGAGATGAACACGGAGGAGGCTGCCAACACCATGGTGAACTACTACACC TCGGTGACCCCTGTGCTGCGCGGCCAGCCCATCTACATCCAGTTCTCCAACCACAAGGAGCTGAAGACC GACAGCTCTCCCAACCAGGCGCGGGCCCAGGCGGCCCTGCAGGCGGTGAACTCGGTCCAGTCGGGGAAC CTGGCCTTGGCTGCCTCGGCGGCGGCCGTGGACGCAGGGATGGCGATGGCCGGGCAGAGCCCCGTGCTC AGGATCATCGTGGAGAACCTCTTCTACCCTGTGACCCTGGATGTGCTGCACCAGATTTTCTCCAAGTTC GGCACAGTGTTGAAGATCATCACCTTCACCAAGAACAACCAGTTCCAGGCCCTGCTGCAGTATGCGGAC CCCGTGAGCGCCCAGCACGCCAAGCTGTCGCTGGACGGGCAGAACATCTACAACGCCTGCTGCACGCTG CGCATCGACTTTTCCAAGCTCACCAGCCTCAACGTCAAGTACAACAATGACAAGAGCCGTGACTACACA CGCCCAGACCTGCCTTCCGGGGACAGCCAGCCCTCGCTGGACCAGACCATGGCCGCGGCCTTCGGTGCA CCTGGTATAATCTCAGCCTCTCCGTATGCAGGAGCTGGTTTCCCTCCCACCTTTGCCATTCCTCAAGCT GCAGGCCTTTCCGTTCCGAACGTCCACGGCGCCCTGGCCCCCCTGGCCATCCCCTCGGCGGCGGCGGCA GCTGCGGCGGCAGGTCGGATCGCCATCCCGGGCCTGGCGGGGGCAGGAAATTCTGTATTGCTGGTCAGC AACCTCAACCCAGAGAGAGTCACACCCCAAAGCCTCTTTATTCTTTTCGGCGTCTACGGTGACGTGCAG CGCGTGAAGATCCTGTTCAATAAGAAGGAGAACGCCCTAGTGCAGATGGCGGACGGCAACCAGGCCCAG CTGGCCATGAGCCACCTGAACGGGCACAAGCTGCACGGGAAGCCCATCCGCATCACGCTCTCGAAGCAC CAGAACGTGCAGCTGCCCCGCGAGGGCCAGGAGGACCAGGGCCTGACCAAGGACTACGGCAACTCACCC CTGCACCGCTTCAAGAAGCCGGGCTCCAAGAACTTCCAGAACATATTCCCGCCCTCGGCCACGCTGCAC CTCTCCAACATCCCGCCCTCAGTCTCCGAGGAGGATCTCAAGGTCCTGTTTTCCAGCAATGGGGGCGTC GTCAAAGGATTCAAGTTCTTCCAGAAGGACCGCAAGATGGCACTGATCCAGATGGGCTCCGTGGAGGAG GCGGTCCAGGCCCTCATTGACCTGCACAACCACGACCTCGGGGAGAACCACCACCTGCGGGTCTCCTTC TCCAAGTCCACCATCTAG (SEQ ID NO: 32) PTTG1IP Entrez ID: 754; OMIM: 603784; Uniprot ID: PTTG_HUMAN; ENSEMBL ID: ENSG00000183255 ATGGCGCCCGGAGTGGCCCGCGGGCCGACGCCGTACTGGAGGTTGCGCCTCGGTGGCGCCGCGCTGCTC CTGCTGCTCATCCCGGTGGCCGCCGCGCAGGAGCCTCCCGGAGCTGCTTGTTCTCAGAACACAAACAAA ACCTGTGAAGAGTGCCTGAAGAACGTCTCCTGTCTTTGGTGCAACACTAACAAGGCTTGTCTGGACTAC CCAGTTACAAGCGTCTTGCCACCGGCTTCCCTTTGTAAATTGAGCTCTGCACGCTGGGGAGTTTGTTGG GTGAACTTTGAGGCGCTGATCATCACCATGTCGGTAGTCGGGGGAACCCTCCTCCTGGGCATTGCCATC TGCTGCTGCTGCTGCTGCAGGAGGAAGAGGAGCCGGAAGCCGGACAGGAGTGAGGAGAAGGCCATGCGT GAGCGGGAGGAGAGGCGGATACGGCAGGAGGAACGGAGAGCAGAGATGAAGACAAGACATGATGAAATC AGAAAAAAATATGGCCTGTTTAAAGAAGAAAACCCGTATGCTAGATTTGAAAACAACTAA (SEQ ID NO: 33) RHBDF2 Entrez ID: 79651; OMIM:; Uniprot ID: RHDF2_HUMAN; ENSEMBL ID:  ENSG00000129667 ATGGCCTCTGCTGACAAGAATGGCGGGAGCGTGTCCTCTGTGTCCAGCAGCCGCCTGCAGAGCCGGAAG CCACCCAACCTCTCCATCACCATCCCGCCACCCGAGAAAGAGACCCAGGCCCCTGGCGAGCAGGACAGC ATGCTGCCTGAGAGGAAGAACCCAGCCTACTTGAAGAGCGTCAGCCTCCAGGAGCCACGCAGCCGATGG CAGGAGAGTTCAGAGAAGCGCCCTGGCTTCCGCCGCCAGGCCTCACTGTCCCAGAGCATCCGCAAGGGC GCAGCCCAGTGGTTTGGAGTCAGCGGCGACTGGGAGGGGCAGCGGCAGCAGTGGCAGCGCCGCAGCCTG CACCACTGCAGCATGCGCTACGGCCGCCTGAAGGCCTCGTGCCAGCGTGACCTGGAGCTCCCCAGCCAG GAGGCACCGTCCTTCCAGGGCACTGAGTCCCCAAAGCCCTGCAAGATGCCCAAGATTGTGGATCCGCTG GCCCGGGGCCGGGCCTTCCGCCACCCGGAGGAGATGGACAGGCCCCACGCCCCGCACCCACCGCTGACC CCCGGAGTCCTGTCCCTCACCTCCTTCACCAGTGTCCGTTCTGGCTACTCCCACCTGCCACGCCGCAAG AGAATGTCTGTGGCCCACATGAGCTTGCAAGCTGCCGCTGCCCTCCTCAAGGGGCGCTCGGTGCTGGAT GCCACCGGACAGCGGTGCCGGGTGGTCAAGCGCAGCTTTGCCTTCCCGAGCTTCCTGGAGGAGGATGTG GTCGATGGGGCAGACACGTTTGACTCCTCCTTTTTTAGTAAGGAAGAAATGAGCTCCATGCCTGATGAT GTCTTTGAGTCCCCCCCACTCTCTGCCAGCTACTTCCGAGGGATCCCACACTCAGCCTCCCCTGTCTCC CCCGATGGGGTGCAAATCCCTCTGAAGGAGTATGGCCGAGCCCCAGTCCCCGGGCCCCGGCGCGGCAAG CGCATCGCCTCCAAGGTGAAGCACTTTGCCTTTGATCGGAAGAAGCGGCACTACGGCCTCGGCGTGGTG GGCAACTGGCTGAACCGCAGCTACCGCCGCAGCATCAGCAGCACTGTGCAGCGGCAGCTGGAGAGCTTC GACAGCCACCGGCCCTACTTCACCTACTGGCTGACCTTCGTCCATGTCATCATCACGCTGCTGGTGATT TGCACGTATGGCATCGCACCCGTGGGCTTTGCCCAGCACGTCACCACCCAGCTGGTGCTGCGGAACAAA GGTGTGTACGAGAGCGTGAAGTACATCCAGCAGGAGAACTTCTGGGTTGGCCCCAGCTCGATTGACCTG ATCCACCTGGGGGCCAAGTTCTCACCCTGCATCCGGAAGGACGGGCAGATCGAGCAGCTGGTGCTGCGC GAGCGAGACCTGGAGCGGGACTCAGGCTGCTGTGTCCAGAATGACCACTCCGGATGCATCCAGACCCAG CGGAAGGACTGCTCGGAGACTTTGGCCACTTTTGTCAAGTGGCAGGATGACACTGGGCCCCCCATGGAC AAGTCTGATCTGGGCCAGAAGCGGACTTCGGGGGCTGTCTGCCACCAGGACCCCAGGACCTGCGAGGAG CCAGCCTCCAGCGGTGCCCACATCTGGCCCGATGACATCACTAAGTGGCCGATCTGCACAGAGCAGGCC AGGAGCAACCACACAGGCTTCCTGCACATGGACTGCGAGATCAAGGGCCGCCCCTGCTGCATCGGCACC AAGGGCAGCTGTGAGATCACCACCCGGGAATACTGTGAGTTCATGCACGGCTATTTCCATGAGGAAGCA ACACTCTGCTCCCAGGTGCACTGCTTGGACAAGGTGTGTGGGCTGCTGCCCTTCCTCAACCCTGAGGTC CCAGATCAGTTCTACAGGCTCTGGCTGTCTCTCTTCCTACATGCTGGCGTGGTGCACTGCCTCGTGTCT GTGGTCTTTCAAATGACCATCCTGAGGGACCTGGAGAAGCTGGCCGGCTGGCACCGTATCGCCATCATC TTCATCCTCAGTGGCATCACAGGCAACCTCGCCAGTGCCATCTTTCTCCCATACCGGGCAGAGGTGGGC CCGGCCGGCTCACAGTTCGGCCTCCTCGCCTGCCTCTTCGTGGAGCTCTTCCAGAGCTGGCCGCTGCTG GAGAGGCCCTGGAAGGCCTTCCTCAACCTCTCGGCCATCGTGCTCTTCCTGTTCATCTGTGGCCTCCTG CCCTGGATCGACAACATCGCCCACATCTTCGGCTTCCTCAGTGGCCTGCTGCTGGCCTTCGCCTTCCTG CCCTACATCACCTTCGGCACCAGCGACAAGTACCGCAAGCGGGCACTCATCCTGGTGTCACTGCTGGCC TTTGCCGGCCTCTTCGCCGCCCTCGTGCTGTGGCTGTACATCTACCCCATTAACTGGCCCTGGATCGAG CACCTCACCTGCTTCCCCTTCACCAGCCGCTTCTGCGAGAAGTATGAGCTGGACCAGGTGCTGCACTGA (SEQ ID NO: 34) SERTAD1 Entrez ID: 29950; OMIM:; Uniprot ID: SRTD1_HUMAN; ENSEMBL ID:  ENSG00000197019 ATGCTGAGCAAGGGTCTGAAGCGGAAACGGGAGGAGGAGGAGGAGAAGGAACCTCTGGCAGTCGACTCC TGGTGGCTAGATCCTGGCCACACAGCGGTGGCACAGGCACCCCCGGCCGTGGCCTCTAGCTCCCTCTTT GACCTCTCAGTGCTCAAGCTCCACCACAGCCTGCAGCAGAGTGAGCCGGACCTGCGGCACCTGGTGCTG GTCGTGAACACTCTGCGGCGCATCCAGGCGTCCATGGCACCCGCGGCTGCCCTGCCACCTGTGCCTAGC CCACCTGCAGCCCCCAGTGTGGCTGACAACTTACTGGCAAGCTCGGACGCTGCCCTTTCAGCCTCCATG GCCAGCCTCCTGGAGGACCTCAGCCACATTGAGGGCCTGAGTCAGGCTCCCCAACCCTTGGCAGACGAG GGGCCACCAGGCCGTAGCATCGGGGGAGCAGCGCCCAGCCTGGGTGCCTTGGACCTGCTGGGCCCAGCC ACTGGCTGTCTACTGGACGATGGGCTTGAGGGCCTGTTTGAGGATATTGACACCTCTATGTATGACAAT GAACTTTGGGCACCAGCCTCTGAGGGCCTCAAACCAGGCCCTGAGGATGGGCCGGGCAAGGAGGAAGCT CCGGAGCTGGACGAGGCCGAATTGGACTACCTCATGGATGTGCTGGTGGGCACACAGGCACTGGAGCGA CCGCCGGGGCCAGGGCGCTGA (SEQ ID NO: 35) SLC2A5 Entrez ID: 6518; OMIM: 138230; Uniprot ID: GTR5_HUMAN; ENSEMBL ID: ENSG00000142583 ATGGAGCAACAGGATCAGAGCATGAAGGAAGGGAGGCTGACGCTTGTGCTTGCCCTGGCAACCCTGATA GCTGCCTTTGGGTCATCCTTCCAGTATGGGTACAACGTGGCTGCTGTCAACTCCCCAGCACTGCTCATG CAACAATTTTACAATGAGACTTACTATGGTAGGACCGGTGAATTCATGGAAGACTTCCCCTTGACGTTG CTGTGGTCTGTAACCGTGTCCATGTTTCCATTTGGAGGGTTTATCGGATCCCTCCTGGTCGGCCCCTTG GTGAATAAATTTGGCAGAAAAGGGGCCTTGCTGTTCAACAACATATTTTCTATCGTGCCTGCGATCTTA ATGGGATGCAGCAGAGTCGCCACATCATTTGAGCTTATCATTATTTCCAGACTTTTGGTGGGAATATGT GCAGGTGTATCTTCCAACGTGGTCCCCATGTACTTAGGGGAGCTGGCCCCTAAAAACCTGCGGGGGGCT CTCGGGGTGGTGCCCCAGCTCTTCATCACTGTTGGCATCCTTGTGGCCCAGATCTTTGGTCTTCGGAAT CTCCTTGCAAACGTAGATGGTGAGTTCAGGACATCTCGGGAGCACCCCCACCCCTTCACCACTACCCTT GGCCCCCTCCTTGTGTTCCAAAGCCACCACCACAGGACAGGACTTTCTGCAGACTGGTCTCTTCTAACA GGCTGGATGTCCTTGGGGGGCCCATCCTGTCCCGAGCCAACATAG (SEQ ID NO: 36) STAMBPL1 Entrez ID: 57559; OMIM: 612352; Uniprot ID: STALP_HUMAN; ENSEMBL ID: ENSG00000138134 ATGGATCAGCCTTTTACTGTGAATTCTCTGAAAAAGTTAGCTGCTATGCCTGACCATACAGATGTTTCC CTAAGCCCAGAAGAGCGAGTCCGTGCCCTAAGCAAGCTTGGTTGTAATATCACCATCAGTGAAGACATC ACTCCACGACGTTACTTTAGGTCTGGAGTAGAGATGGAGAGGATGGCGTCTGTGTATTTGGAAGAAGGA AATTTGGAAAATGCCTTTGTTCTTTATAATAAATTTATAACCTTATTTGTAGAAAAGCTTCCTAACCAT CGAGATTACCAGCAATGTGCAGTACCTGAAAAGCAGGATATTATGAAGAAACTGAAGGAGATTGCATTC CCAAGGACAGATGAATTGAAAAACGACCTTTTAAAGAAATATAACGTAGAATACCAAGAATATTTGCAA AGCAAAAACAAATATAAAGCTGAAATTCTCAAAAAATTGGAGCATCAGAGATTGATAGAGGCAGAAAGG AAGCGGATTGCTCAGATGCGCCAGCAGCAGCTAGAATCGGAGCAGTTTCTGTTTTTCGAAGATCAACTC AAGAAGCAAGAGTTAGCCCGAGGTCAAATGCGAAGTCAGCAAACCTCAGGGCTGTCAGAGCAGATTGAT GGGAGCGCTTTGTCCTGCTTTTCCACACACCAGAACAATTCCTTGCTGAATGTATTTGCAGATCAACCT AATAAAAGTGATGCAACCAATTATGCTAGCCACTCTCCTCCTGTAAACAGGGCCTTAACACCAGCTGCT ACTCTAAGTGCTGTTCAGAATTTAGTGGTTGAAGGACTGCGATGTGTAGTTTTGCCAGAAGATCTTTGC CACAAATTTCTGCAACTGGCAGAATCTAATACAGTGAGAGGAATAGAAACCTGTGGAATACTCTGTGGA AAACTGACACATAATGAATTTACTATTACCCATGTAATTGTGCCAAAGCAGTCTGCGGGACCAGACTAT TGTGACATGGAGAATGTAGAGGAATTATTCAATGTTCAGGATCAACATGATCTCCTCACTCTAGGATGG ATCCATACACATCCCACTCAAACTGCATTTTTATCCAGCGTTGATCTTCACACTCACTGTTCCTATCAA CTCATGTTGCCAGAGGCCATTGCCATTGTTTGCTCACCAAAGCATAAAGACACTGGCATCTTCAGGCTC ACCAATGCTGGCATGCTTGAGGTTTCTGCTTGTAAAAAAAAGGGCTTTCATCCACACACCAAGGAGCCC AGGCTGTTCAGTATATGCAAACATGTGTTGGTAAAAGACATAAAAATAATTGTGTTGGATCTGAGGTGA (SEQ ID NO: 37) TESC Entrez ID: 54997; OMIM: 611585; Uniprot ID: TESC_HUMAN; ENSEMBL ID: ENSG00000088992 ATGGGCGCTGCCCACTCCGCGTCTGAGGAGGTGCGGGAGCTCGAGGGCAAGACCGGCTTCTCATCGGAT CAGATCGAGCAGCTCCATCGGAGATTTAAGCAGCTGAGTGGAGATCAGCCTACCATTCGGAACCTGCGC AAGGGACCCAGTGGCCTGGCTGATGAGATCAATTTCGAGGACTTCCTGACCATCATGTCCTACTTCCGG CCCATCGACACCACCATGGACGAGGAACAGGTGGAGCTGTCCCGGAAGGAGAAGCTGAGATTTCTGTTC CACATGTACGACTCGGACAGCGACGGCCGCATCACTCTGGAAGAATATCGAAATGTGGTCGAGGAGCTG CTGTCGGGAAACCCTCACATCGAGAAGGAGTCCGCTCGCTCCATCGCCGACGGGGCCATGATGGAGGCG GCCAGCGTGTGCATGGGGCAGATGGAGCCTGATCAGGTGTACGAGGGGATCACCTTCGAGGACTTCCTG AAGATCTGGCAGGGGATCGACATTGAGACCAAGATGCACGTCCGCTTCCTTAACATGGAAACCATGGCC CTCTGCCACTGA (SEQ ID NO: 38) TIMP1 Entrez ID: 7076; OMIM: 305370; Uniprot ID: TIMP1_HUMAN; ENSEMBL ID: ENSG00000102265 ATGGCCCCCTTTGAGCCCCTGGCTTCTGGCATCCTGTTGTTGCTGTGGCTGATAGCCCCCAGCAGGGCC TGCACCTGTGTCCCACCCCACCCACAGACGGCCTTCTGCAATTCCGACCTCGTCATCAGGGCCAAGTTC GTGGGGACACCAGAAGTCAACCAGACCACCTTATACCAGCGTTATGAGATCAAGATGACCAAGATGTAT AAAGGGTTCCAAGCCTTAGGGGATGCCGCTGACATCCGGTTCGTCTACACCCCCGCCATGGAGAGTGTC TGCGGATACTTCCACAGGTCCCACAACCGCAGCGAGGAGTTTCTCATTGCTGGAAAACTGCAGGATGGA CTCTTGCACATCACTACCTGCAGTTTTGTGGCTCCCTGGAACAGCCTGAGCTTAGCTCAGCGCCGGGGC TTCACCAAGACCTACACTGTTGGCTGTGAGGAATGCACAGTGTTTCCCTGTTTATCCATCCCCTGCAAA CTGCAGAGTGGCACTCATTGCTTGTGGACGGACCAGCTCCTCCAAGGCTCTGAAAAGGGCTTCCAGTCC CGTCACCTTGCCTGCCTGCCTCGGGAGCCAGGGCTGTGCACCTGGCAGTCCCTGCGGTCCCAGATAGCC TGA (SEQ ID NO: 39) TNPO1 Entrez ID: 3842; OMIM: 602901; Uniprot ID: TNPO1_HUMAN; ENSEMBL ID: ENSG00000083312 ATGGTGTGGGACCGGCAAACCAAGATGGAGTATGAGTGGAAACCTGACGAGCAAGGGCTTCAGCAAATC CTGCAGCTGTTGAAGGAGTCCCAGTCCCCAGACACCACCATCCAGAGAACCGTGCAACAAAAACTGGAA CAACTTAATCAGTATCCAGACTTTAACAACTACTTGATTTTTGTTCTTACAAAATTAAAATCTGAAGAT GAACCCACAAGATCATTGAGTGGTCTTATCTTGAAGAATAATGTGAAAGCACACTTTCAGAACTTCCCA AATGGTGTAACAGACTTTATTAAAAGTGAATGTTTAAATAATATTGGTGACTCCTCTCCTCTGATTAGA GCCACTGTTGGTATTTTGATCACAACTATAGCCTCCAAGGGAGAATTGCAGAATTGGCCTGACCTCTTA CCAAAACTCTGTAGCCTGTTGGATTCTGAAGATTATAATACCTGTGAGGGAGCATTTGGTGCCCTTCAG AAGATTTGTGAAGATTCTGCTGAGATTTTAGACAGTGATGTTTTAGATCGTCCTCTCAACATCATGATT CCCAAATTTTTACAGTTCTTCAAGCATAGTAGTCCAAAAATAAGGTCTCACGCTGTTGCATGTGTCAAT CAGTTTATCATCAGTAGGACTCAAGCTCTAATGTTGCACATTGATTCTTTTATTGAGAATCTCTTTGCA TTAGCTGGTGATGAAGAACCAGAGGTACGGAAAAATGTGTGCCGAGCACTTGTGATGTTGCTCGAAGTT CGAATGGATCGCCTGCTTCCTCACATGCATAATATAGTTGAGTACATGCTACAGAGGACTCAAGATCAA GATGAAAATGTGGCTTTAGAAGCCTGTGAATTTTGGCTAACTTTAGCTGAACAGCCAATATGCAAAGAT GTACTCGTAAGGCATCTTCCTAAGTTGATTCCTGTGTTAGTGAATGGCATGAAGTACTCAGACATAGAT ATTATCCTACTTAAGGGTGATGTTGAAGAAGACGAAACGATTCCTGATAGTGAACAGGATATACGGCCA CGTTTTCACCGATCGAGGACGGTGGCTCAGCAGCATGATGAAGATGGAATTGAAGAGGAAGATGATGAT GATGATGAAATTGATGATGATGATACAATTTCTGACTGGAATCTAAGAAAATGTTCTGCTGCTGCCCTG GATGTTCTTGCAAATGTGTATCGTGATGAACTGCTGCCACATATTTTGCCCCTTTTGAAAGAATTACTT TTTCATCATGAATGGGTTGTTAAAGAATCAGGCATTTTGGTTTTAGGAGCAATTGCTGAAGGTTGCATG CAGGGCATGATTCCATACTTGCCTGAGCTTATTCCTCACCTTATTCAGTGCCTCTCTGATAAAAAGGCT CTTGTGCGTTCCATAACATGCTGGACTCTTAGCCGCTATGCACACTGGGTGGTCAGCCAGCCGCCAGAC ACGTACCTGAAGCCATTAATGACAGAATTGCTAAAGCGCATCCTGGACAGCAACAAGAGAGTACAAGAA GCTGCCTGCAGTGCCTTTGCTACCCTAGAAGAGGAGGCTTGTACAGAACTTGTTCCTTACCTTGCTTAT ATACTTGATACCCTGGTCTTTGCATTTAGTAAATACCAGCATAAGAACCTGCTCATTCTTTACGATGCC ATAGGAACATTAGCAGATTCAGTAGGACATCATTTAAACAAACCAGAATATATTCAGATGCTAATGCCT CCACTGATCCAGAAATGGAACATGTTAAAGGATGAAGATAAAGATCTCTTCCCTTTACTTGAGTGCCTA TCTTCAGTTGCCACAGCACTGCAGTCTGGATTCCTTCCGTACTGTGAACCTGTGTATCAGCGTTGTGTA AACCTAGTACAGAAGACTCTTGCACAAGCCATGCTAAACAATGCTCAACCAGATCAATATGAAGCTCCA GATAAAGATTTTATGATAGTGGCTCTTGATTTACTGAGTGGCCTGGCTGAAGGACTTGGAGGCAACATT GAACAGCTGGTAGCCCGAAGTAACATCCTGACACTAATGTATCAGTGCATGCAGGATAAAATGCCAGAA GTTCGACAGAGTTCTTTTGCCCTGTTAGGTGACCTCACAAAAGCTTGCTTTCAGCATGTTAAGCCTTGT ATAGCTGATTTCATGCCAATATTGGGAACCAACCTAAATCCAGAATTCATTTCAGTCTGCAACAATGCC ACATGGGCAATTGGAGAAATCTCCATTCAAATGGGTATAGAGATGCAGCCTTATATTCCTATGGTGTTG CACCAGCTTGTAGAAATCATTAACAGACCCAACACACCAAAGACGTTGTTAGAGAATACAGCAATAACA ATTGGTCGTCTTGGTTACGTTTGTCCTCAAGAGGTGGCCCCCATGCTACAGCAGTTTATAAGACCCTGG TGCACCTCTCTGAGAAACATAAGAGACAATGAGGAAAAGGATTCAGCATTCCGTGGAATTTGTACCATG ATCAGTGTGAATCCCAGTGGCGTAATCCAAGATTTTATATTTTTTTGTGATGCCGTTGCATCATGGATT AACCCAAAAGATGATCTCAGAGACATGTTCTGTAAGATCCTTCATGGATTTAAAAATCAAGTTGGCGAT GAAAATTGGAGGCGTTTCTCTGACCAGTTTCCTCTTCCCTTAAAAGAGCGTCTTGCAGCTTTTTATGGT GTTTAA (SEQ ID NO: 40) TXNIP Entrez ID: 10628; OMIM: 606599; Uniprot ID: TXNIP_HUMAN; ENSEMBL ID: ENSG00000117289 ATGGTGATGTTCAAGAAGATCAAGTCTTTTGAGGTGGTCTTTAACGACCCTGAAAAGGTGTACGGCAGT GGCGAGAAGGTGGCTGGCCGGGTGATAGTGGAGGTGTGTGAAGTTACTCGTGTCAAAGCCGTTAGGATC CTGGCTTGCGGAGTGGCTAAAGTGCTTTGGATGCAGGGATCCCAGCAGTGCAAACAGACTTCGGAGTAC CTGCGCTATGAAGACACGCTTCTTCTGGAAGACCAGCCAACAGGTGAGAATGAGATGGTGATCATGAGA CCTGGAAACAAATATGAGTACAAGTTCGGCTTTGAGCTTCCTCAGGGGCCTCTGGGAACATCCTTCAAA GGAAAATATGGGTGTGTAGACTACTGGGTGAAGGCTTTTCTTGACCGCCCGAGCCAGCCAACTCAAGAG ACAAAGAAAAACTTTGAAGTAGTGGATCTGGTGGATGTCAATACCCCTGATTTAATGGCACCTGTGTCT GCTAAAAAAGAAAAGAAAGTTTCCTGCATGTTCATTCCTGATGGGCGGGTGTCTGTCTCTGCTCGAATT GACAGAAAAGGATTCTGTGAAGGTGATGAGATTTCCATCCATGCTGACTTTGAGAATACATGTTCCCGA ATTGTGGTCCCCAAAGCTGCCATTGTGGCCCGCCACACTTACCTTGCCAATGGCCAGACCAAGGTGCTG ACTCAGAAGTTGTCATCAGTCAGAGGCAATCATATTATCTCAGGGACATGCGCATCATGGCGTGGCAAG AGCCTTCGGGTTCAGAAGATCAGGCCTTCTATCCTGGGCTGCAACATCCTTCGAGTTGAATATTCCTTA CTGATCTATGTTAGCGTTCCTGGATCCAAGAAGGTCATCCTTGACCTGCCCCTGGTAATTGGCAGCAGA TCAGGTCTAAGCAGCAGAACATCCAGCATGGCCAGCCGAACCAGCTCTGAGATGAGTTGGGTAGATCTG AACATCCCTGATACCCCAGAAGCTCCTCCCTGCTATATGGATGTCATTCCTGAAGATCACCGATTGGAG AGCCCAACCACTCCTCTGCTAGATGACATGGATGGCTCTCAAGACAGCCCTATCTTTATGTATGCCCCT GAGTTCAAGTTCATGCCACCACCGACTTATACTGAGGTGGATCCCTGCATCCTCAACAACAATGTGCAG TGA (SEQ ID NO: 41) VAMP1 Entrez ID: 6843; OMIM: 185880; Uniprot ID: VAMP1_HUMAN; ENSEMBL ID: ENSG00000139190 ATGTCTGCTCCAGCTCAGCCACCTGCTGAAGGGACAGAAGGGACTGCCCCAGGTGGGGGTCCCCCTGGC CCTCCTCCTAACATGACCAGTAACAGACGACTACAGCAAACCCAGGCACAAGTGGAGGAGGTGGTGGAC ATCATACGTGTGAACGTGGACAAGGTCCTGGAGAGGGACCAGAAGCTGTCAGAGCTGGATGACCGAGCT GATGCCTTGCAGGCAGGAGCATCACAATTTGAGAGCAGTGCTGCCAAGCTAAAGAGGAAGTATTGGTGG AAAAACTGCAAGATGATGATCATGCTGGGAGCCATCTGTGCCATCATCGTGGTAGTTATTGTAATCTAC TTTTTTACTTGA (SEQ ID NO: 42) VHL Entrez ID: 7428; OMIM: 608537; Uniprot ID: VHL_HUMAN; ENSEMBL ID:  ENSG00000134086  ATGCCCCGGAGGGCGGAGAACTGGGACGAGGCCGAGGTAGGCGCGGAGGAGGCAGGCGTCGAAGAGTAC GGCCCTGAAGAAGACGGCGGGGAGGAGTCGGGCGCCGAGGAGTCCGGCCCGGAAGAGTCCGGCCCGGAG GAACTGGGCGCCGAGGAGGAGATGGAGGCCGGGCGGCCGCGGCCCGTGCTGCGCTCGGTGAACTCGCGC GAGCCCTCCCAGGTCATCTTCTGCAATCGCAGTCCGCGCGTCGTGCTGCCCGTATGGCTCAACTTCGAC GGCGAGCCGCAGCCCTACCCAACGCTGCCGCCTGGCACGGGCCGCCGCATCCACAGCTACCGAGGTCAC CTTTGGCTCTTCAGAGATGCAGGGACACACGATGGGCTTCTGGTTAACCAAACTGAATTATTTGTGCCA TCTCTCAATGTTGACGGACAGCCTATTTTTGCCAATATCACACTGCCAGTGTATACTCTGAAAGAGCGA TGCCTCCAGGTTGTCCGGAGCCTAGTCAAGCCTGAGAATTACAGGAGACTGGACATCGTCAGGTCGCTC TACGAAGATCTGGAAGACCACCCAAATGTGCAGAAAGACCTGGAGCGGCTGACACAGGAGCGCATTGCA CATCAACGGATGGGAGATTGA (SEQ ID NO: 43) ZFP36L1 Entrez ID: 677; OMIM: 601064; Uniprot ID: TISB_HUMAN; ENSEMBL ID: ENSG00000185650  ATGACCACCACCCTCGTGTCTGCCACCATCTTCGACTTGAGCGAAGTTTTATGCAAGGGTAACAAGATG CTCAACTATAGTGCTCCCAGTGCAGGGGGTTGCCTGCTGGACAGAAAGGCAGTGGGCACCCCTGCTGGT GGGGGCTTCCCTCGGAGGCACTCAGTCACCCTGCCCAGCTCCAAGTTCCACCAGAACCAGCTCCTCAGC AGCCTCAAGGGTGAGCCAGCCCCCGCTCTGAGCTCGCGAGACAGCCGCTTCCGAGACCGCTCCTTCTCG GAAGGGGGCGAGCGGCTGCTGCCCACCCAGAAGCAGCCCGGGGGCGGCCAGGTCAACTCCAGCCGCTAC AAGACGGAGCTGTGCCGCCCCTTTGAGGAAAACGGTGCCTGTAAGTACGGGGACAAGTGCCAGTTCGCA CACGGCATCCACGAGCTCCGCAGCCTGACCCGCCACCCCAAGTACAAGACGGAGCTGTGCCGCACCTTC CACACCATCGGCTTTTGCCCCTACGGGCCCCGCTGCCACTTCATCCACAACGCTGAAGAGCGCCGTGCC CTGGCCGGGGCCCGGGACCTCTCCGCTGACCGTCCCCGCCTCCAGCATAGCTTTAGCTTTGCTGGGTTT CCCAGTGCCGCTGCCACCGCCGCTGCCACCGGGCTGCTGGACAGCCCCACGTCCATCACCCCACCCCCT ATTCTGAGCGCCGATGACCTCCTGGGCTCACCTACCCTGCCCGATGGCACCAATAACCCTTTTGCCTTC TCCAGCCAGGAGCTGGCAAGCCTCTTTGCCCCTAGCATGGGGCTGCCCGGGGGTGGCTCCCCGACCACC TTCCTCTTCCGGCCCATGTCCGAGTCCCCTCACATGTTTGACTCTCCCCCCAGCCCTCAGGATTCTCTC TCGGACCAGGAGGGCTACCTGAGCAGCTCCAGCAGCAGCCACAGTGGCTCAGACTCCCCGACCTTGGAC AACTCAAGACGCCTGCCCATCTTCAGCAGACTTTCCATCTCAGATGACTAA (SEQ ID NO: 44) As is known in the art, individuals diagnosed with autism shown dysregulated gene expression in leukocytes (see, e.g. Nishimura et al., Human Molecular Genetics 2007 16 (14): 1682-1698). Moreover, gene ontology enrichment analysis showed that genes upregulated in autistic cortex were enriched for gene ontology categories implicated in immune and inflammatory response. Genes including those identified in Table B were shown to have overlapping expression patterns with brain and blood cells in one or more data sets, data supporting their utility as peripheral biomarkers.

The present invention is not to be limited in scope by the embodiments disclosed herein, which are intended as single illustrations of individual aspects of the invention, and any that are functionally equivalent are within the scope of the invention. Various modifications to the models and methods of the invention, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description and teachings, and are similarly intended to fall within the scope of the invention. Such modifications or other embodiments can be practiced without departing from the true scope and spirit of the invention. 

1. A method of identifying a test mammalian cell having a gene expression profile observed in individuals diagnosed with autism comprising: observing an expression profile of at least one gene comprising a sequence selected from the group consisting of SEQ ID NOs: 1-44 in the test mammalian cell; wherein an expression profile of a gene in the group that is at least two standard deviations from a mean expression profile of the gene in a control mammalian cell obtained from an individual not affected with autism identifies the test mammalian cell as having a gene expression profile observed in individuals diagnosed with autism.
 2. The method of claim 1, wherein the expression of a gene in the group is at least three, four or five standard deviations from the mean expression of the gene observed in a control mammalian cell.
 3. The method of claim 1, wherein the expression level of a gene in the group is at least 20, 30, 40, 50, 60 or 70% above or below the expression level of the gene observed in the control mammalian cell.
 4. The method of claim 1, wherein mRNA expression is observed.
 5. The method of claim 4, wherein the expression profile is observed using: (a) a microarray of polynucleotides; and/or (b) a quantitative PCR (qPCR) process.
 6. The method of claim 1, wherein an expression profile of at least, 2, 3, 4, 5, 6, 7, 8, 9 or 10 genes in the group is observed.
 7. The method of claim 1, wherein the expression profile of the test mammalian cell is observed using a computer system comprising a processor element and a memory storage element adapted to process and store data from one or more expression profiles.
 8. The method of claim 1, wherein the test mammalian cell or the control mammalian cell is a leukocyte obtained from the peripheral blood.
 9. The method of claim 1, wherein the test mammalian cell is obtained from an individual previously identified as exhibiting restricted repetitive behaviors or speech delay.
 10. The method of claim 1, wherein the control mammalian cell is obtained from an individual previously identified as not exhibiting restricted repetitive behaviors or speech delay.
 11. The method of claim 1, wherein the test mammalian cell and the control mammalian cell are obtained from individuals who are related as siblings or as a parent and a child.
 12. The method of claim 1, wherein the test mammalian cell is obtained from an individual identified as having a family member previously identified as exhibiting restricted repetitive behaviors or speech delay.
 13. The method of claim 1, wherein the test mammalian cell is obtained from an individual where at least one evaluation is performed from a diagnostic procedure for autism comprising: (a) a Autism Diagnostic Interview (ADI-R); (b) an Autism Diagnostic Observation Schedule (ADOS); (c) an IQ surrogate test based on Raven's Progressive Matrices; or (d) observations of restricted repetitive behaviors or speech delay.
 14. The method of claim 13, wherein the at least one evaluation is performed prior to observing the expression profile of the at least one gene.
 15. A kit comprising: a container; a primer composition contained within said container, wherein the primer composition includes a polymerase chain reaction (PCR) primer effective in the quantitative real time analysis of the mRNA expression levels of one or more genes selected from the group consisting of SEQ ID NOs: 1-44; and a buffer composition.
 16. The kit of claim 15, wherein the kit comprises a plurality of chain reaction (PCR) primers effective in the quantitative real time analysis of the mRNA expression levels of different genes selected from the group consisting of SEQ ID NOs: 1-44.
 17. The kit of claim 15, further comprising a computer readable a memory storage element adapted to process and store data from one or more expression profiles.
 18. The kit of claim 17, wherein the memory storage element organizes expression profile data into a format adapted for electronic comparisons with a library of expression profile data.
 19. The kit of claim 1, wherein the primer is adapted to bind to mRNA expressed by a human leukocyte.
 20. A method of observing an effect of a compound on an expression profile of at least one gene comprising a sequence selected from the group consisting of SEQ ID NOs: 1-44, the method comprising the steps of: observing an expression profile of the at least one gene in the presence of the compound; and comparing the expression profile that is observed in the presence of the compound with the expression profile that is observed in the absence of the compound; so that the effect of the compound on an expression profile of the at least one gene is observed. 